Chiabotto Giulia, Bruno Stefania, Collino Federica, Camussi Giovanni
Department of Medical Science, University of Torino, Medical School, Torino, Italy.
Department of Molecular Biotechnology and Healthy Science, Molecular Biotechnology Center, University of Torino, Torino, Italy.
PLoS One. 2016 Jul 13;11(7):e0159163. doi: 10.1371/journal.pone.0159163. eCollection 2016.
Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs.
间充质-上皮相互作用在肾小管形态发生及维持肾脏结构中发挥着重要作用。本研究旨在探讨人肾近端小管上皮细胞（RPTECs）产生的细胞外囊泡（EVs）是否可诱导骨髓间充质基质细胞（MSCs）发生间充质-上皮转化。为验证这一假设，我们对EVs的表型和RNA含量进行了表征，并评估了EVs对MSCs的体外摄取及活性。微小RNA（miRNA）分析表明，EVs携带的miR-200家族可能参与了MSCs的上皮定向分化。将骨髓来源的MSCs与EVs、RPTECs来源的全条件培养基或去除EVs的条件培养基共同孵育。作为阳性对照，将MSCs与RPTECs在Transwell系统中共培养。通过实时定量PCR及对特定间充质和上皮标志物的细胞表达进行免疫荧光分析，评估MSCs的上皮定向分化情况。与EVs和全条件培养基孵育一周后，我们观察到MSCs发生了间充质-上皮转化。用去除EVs的条件培养基刺激未引起间充质和上皮基因表达的任何变化。由于发现EVs含有miR-200家族，我们用合成的miR-200模拟物转染MSCs。转染一周后，MSCs发生了间充质-上皮转化。总之，RPTECs释放的携带miR-200的EVs可诱导MSCs的上皮定向分化，这可能有助于其再生潜能。基于用miR-200模拟物转染MSCs的实验，我们认为miR-200家族可能参与了MSCs的间充质-上皮转化。
