Abstract
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.
Highlights
Mutual interactions between epithelial cells and mesenchymal cells coordinate kidney development, play a pivotal role in maintaining organ integrity in the adult, and contribute to renal regeneration after injury
After 7 days, mRNA levels of specific mesenchymal markers Twist (TWIST1), vimentin (VIM) and FOXC2 were significant reduced in mesenchymal stromal cells (MSCs) (Fig 1A and 1E); MSCs expressed higher levels of the epithelial-specific marker cytokeratin 18 (KRT18), suggesting that Mesenchymal-epithelial transition (MET) occurred in MSCs (Fig 1B and 1H)
To investigate the contribution of paracrine factors in epithelial differentiation of MSCs induced by renal proximal tubular epithelial cells (RPTECs), MSCs were incubated with Conditioned Medium deprived of extracellular vesicles (CM) or with purified extracellular vesicles (EVs)
Summary
Mutual interactions between epithelial cells and mesenchymal cells coordinate kidney development, play a pivotal role in maintaining organ integrity in the adult, and contribute to renal regeneration after injury. The epithelial commitment of bone marrow-derived MSCs induced by renal tubular epithelial cells has been demonstrated in co-culture conditions [1]. Conditioned medium derived from renal tubular epithelial cells has proved to induce an epithelial commitment of adipose-derived adult MSCs [2] and of bone marrow-derived MSCs [3,4]. While there are plenty of studies analyzing EMT related to fibrosis in chronic inflammation and metastasis of tumor [7,8,9,10,11,12,13], relatively little is known about MET associated with kidney formation This process seems to be regulated by genes such as paired box 2 (PAX2), bone morphogenetic protein 7 (BMP7), and Wilms tumor 1 (WT1) [14,15,16]
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