Hyperpigmentation of skin is caused by an imbalance between the melanosome/melanin synthesis in melanocytes and the melanosome/melanin degradation in keratinocytes. Although studies showed that stem cells play a role in hypopigmentation, the underlying mechanisms are far not elucidated. Human amniotic stem cells (hASCs) including human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial stem cells (hAESCs) were considered to be a promising cell source for stem cells-based therapy of many diseases clinically due to their pluripotent potential, no tumorigenesis and immunogenicity, no ethical issues, and potent paracrine effects. Here, we reported that both hASCs and their conditional medium (CM) had a potent anti-hyperpigmentation in skin in vivo and in vitro. hAESCs and hAMSCs were identified by RT-PCR, flow cytometric analysis and immunofluorescence. Effects of hASCs and hASC-CM on pigmentation were evaluated in B16F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH), and mouse ears or human skin substitutes treated with ultraviolet radiation B (UVB). Expressions of the key proteins related with melanogenesis and autophagic flux were detected by western blot in B16F10 cells for further exploring the effects and the underlying mechanisms of hAESC-CM and hAMSC-CM on melanogenesis and melanosome degradation. The hAMSCs exosomes-derived miRNAs were determined by sequencing. RT-PCR, western blot, melanin content analysis and luciferase activity assay were used to determine the hypopigmentation of miR-181a-5p and miR-199a. In our study, we observed that both hASCs and their CM significantly alleviated the α-MSH in B16F10 cells or UVB-induced hyperpigmentation in mouse ears or human skin substitutes by suppressing melanin synthesis and promoting melanosome degradation in vivo and in vitro. Furthermore, we demonstrated that miR-181a-5p and miR-199a derived from hASCs exosomes remarkably inhibited melanogenesis by suppressing MITF (microphthalmia-associated transcription factor) which is a master regulator for governing melanogenesis and promoting melanosome degradation through activating autophagy, respectively. Our studies provided strong evidence that the conditional medium and exosomes derived from hAMSCs inhibit skin hyperpigmentation by suppressing melanogenesis and promoting melanosome degradation, indicating that the hASCs exosomes or their released microRNAs might be as reagents for cell-free therapy in hyperpigmented disorders clinically.

中文翻译：

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人羊膜干细胞来源的外泌体 miR-181a-5p 和 miR-199a 分别抑制皮肤色素沉着过度中的黑色素生成并促进黑色素体降解

皮肤色素沉着过度是由黑素细胞中黑素体/黑色素合成与角质形成细胞中黑素体/黑色素降解之间的不平衡引起的。尽管研究表明干细胞在色素沉着不足中发挥作用，但其潜在机制尚不清楚。人羊膜干细胞（hASCs），包括人羊膜间充质干细胞（hAMSCs）和人羊膜上皮干细胞（hAESCs），由于其多能潜力，被认为是临床上基于干细胞治疗多种疾病的有前景的细胞来源。肿瘤发生和免疫原性，没有伦理问题，并且具有有效的旁分泌作用。在这里，我们报道了 hASC 及其条件培养基 (CM) 在体内和体外都具有有效的抗皮肤色素沉着过度的作用。通过RT-PCR、流式细胞术分析和免疫荧光鉴定hAESCs和hAMSCs。在用 α-促黑素细胞激素 (α-MSH) 刺激的 B16F10 细胞以及用紫外线辐射 B (UVB) 处理的小鼠耳朵或人类皮肤替代物中评估了 hASC 和 hASC-CM 对色素沉着的影响。通过western blot检测B16F10细胞中与黑素生成和自噬流相关的关键蛋白的表达，以进一步探讨hAESC-CM和hAMSC-CM对黑素生成和黑素体降解的影响及其潜在机制。通过测序确定 hAMSC 外泌体衍生的 miRNA。RT-PCR、蛋白质印迹、黑色素含量分析和荧光素酶活性测定用于确定miR-181a-5p和miR-199a的色素沉着不足。在我们的研究中，我们观察到 hASC 及其 CM 通过抑制黑色素合成并促进体内和体外黑素体降解，显着减轻 B16F10 细胞中的 α-MSH 或小鼠耳朵或人类皮肤替代品中 UVB 诱导的色素沉着过度。此外，我们证明源自 hASC 外泌体的 miR-181a-5p 和 miR-199a 分别通过抑制 MITF（小眼相关转录因子）来显着抑制黑素生成，MITF 是控制黑素生成和通过激活自噬促进黑素体降解的主要调节因子。我们的研究提供了强有力的证据，表明hAMSCs衍生的条件培养基和外泌体通过抑制黑素生成和促进黑素体降解来抑制皮肤色素沉着过度，这表明hASCs外泌体或其释放的microRNA可能作为临床色素沉着过度疾病的无细胞治疗的试剂。