Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing
Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing
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DOI：
10.1080/20013078.2019.1565264
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发表时间：
2019-01-01
影响因子：
16
通讯作者：
Kanai, Nobuo
中科院分区：
文献类型：
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作者：
Sjoqvist, Sebastian;Ishikawa, Taichi;Kanai, Nobuo
口腔粘膜表现出独特的再生特性，有时被称为胎儿样伤口愈合。我们研究所的研究人员将口腔黏膜上皮细胞(OMECs)用于再生医学应用，包括角膜置换和食道上皮再生以预防狭窄。在这里，我们从来自健康人体供者(n=8)的临床级Omec片材中分离出外切体，旨在评估外切体刺激上皮再生的临床潜力，并提高对细胞作用模式的理解。从条件培养液(CExo)和非条件培养液(NcExo)中分离外切体。对常见的胞外体标志物进行Western印迹鉴定：CD9和Flotillin为阳性，Annexin V、EpCAM和污染标志物GRP94为阴性。纳米粒子跟踪分析表明，纳米粒子的直径为120 nm，透射电子显微镜显示出相应的尺寸和球状外观。体外培养的人皮肤成纤维细胞呈剂量依赖性增殖抑制，生长因子基因(HGF、VEGFA、FGF2和CTGF)表达显著增加。两组结果相似，但cExo的效果有更大的趋势。为了研究粘附性，将荧光标记的外切体局部应用于猪的食道伤口床，然后清洗。黏附1分钟后即可检测到阳性信号，但黏附时间越长，信号越强。接下来，将标记的外切体添加到大鼠全层皮肤创面，并在应用后5天检测到信号。CExo在第6天和第17天显著减小创面大小。结论：Omec片外切体对皮肤创面愈合具有促进再生的作用。这是首次从外周体的角度研究口腔粘膜的愈合能力。这些发现可能会导致对未来早期食道癌患者进行细胞片和外切体的联合治疗。
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 (n = 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 ex vivo 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.