Sjöqvist Sebastian, Ishikawa Taichi, Shimura Daisuke, Kasai Yoshiyuki, Imafuku Aya, Bou-Ghannam Sophia, Iwata Takanori, Kanai Nobuo
Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden.
Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan.
J Extracell Vesicles. 2019 Jan 20;8(1):1565264. doi: 10.1080/20013078.2019.1565264. eCollection 2019.
The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors ( = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.
口腔黏膜具有独特的再生特性，有时被称为胎儿样伤口愈合。我们研究所的研究人员已将口腔黏膜上皮细胞（OMECs）片用于再生医学应用，包括角膜置换和食管上皮再生以预防狭窄。在此，我们从健康人类供体（n = 8）的临床级OMECs片生产中分离出外泌体，旨在评估外泌体刺激上皮再生的临床潜力，并增进对细胞作用模式的理解。外泌体从条件培养基（cExo）和非条件培养基（ncExo）中分离出来。使用蛋白质免疫印迹法对常见外泌体标志物进行表征：CD9和浮舰蛋白呈阳性，而膜联蛋白V、上皮细胞黏附分子（EpCam）和污染标志物GRP94呈阴性。纳米颗粒跟踪分析显示直径约为120 nm，透射电子显微镜显示出相应的大小和球形外观。暴露于外泌体的人皮肤成纤维细胞显示出增殖的剂量依赖性降低以及生长因子基因表达（HGF、VEGFA、FGF2和CTGF）的显著增加。两组结果相似，但cExo的作用趋势更大。为了研究黏附情况，将荧光标记的外泌体局部应用于猪食管伤口床，随后进行冲洗。黏附仅1分钟后即可检测到阳性信号，但黏附时间延长会产生更强的信号。接下来，将标记的外泌体添加到大鼠的全层皮肤伤口中，应用后长达5天均可检测到信号。cExo在第6天和第17天显著减小了伤口大小。总之，OMECs片的外泌体对皮肤伤口愈合显示出促再生作用。这是首次从外泌体角度研究口腔黏膜的愈合能力。这些发现可能会为未来的早期食管癌患者带来细胞片和外泌体的联合治疗。
