New Insights into Pediatric Kidney Transplant Rejection Biomarkers: Tissue, Plasma and Urine MicroRNAs Compared to Protocol Biopsy Histology.

The early identification of a subclinical rejection (SCR) can improve the long-term outcome of the transplanted kidney through intensified immunosuppression. However, the only approved diagnostic method is the protocol biopsy, which remains an invasive method and not without minor and/or major complications. The protocol biopsy is defined as the sampling of allograft tissue at pre-established times even in the absence of an impaired renal function; however, it does not avoid histological damage. Therefore, the discovery of new possible biomarkers useful in the prevention of SCR has gained great interest. Among all the possible candidates, there are microRNAs (miRNAs), which are short, noncoding RNA sequences, that are involved in mediating numerous post-transcriptional pathways. They can be found not only in tissues, but also in different biological fluids, both as free particles and contained in extracellular vesicles (EVs) released by different cell types. In this study, we firstly performed a retrospective miRNA screening analysis on biopsies and serum EV samples of 20 pediatric transplanted patients, followed by a second screening on another 10 pediatric transplanted patients' urine samples at one year post-transplant. In both cohorts, we divided the patients into two groups: patients with histological SCR and patients without histological SCR at one year post-transplantation. The isolated miRNAs were analyzed in an NGS platform to identify different expressions in the two allograft states. Although no statistical data were found in sera, in the tissue and urinary EVs, we highlighted signatures of miRNAs associated with the histological SCR state.

Extracellular Vesicles Derived from Human Liver Stem Cells Attenuate Chronic Kidney Disease Development in an In Vivo Experimental Model of Renal Ischemia and Reperfusion Injury.

The potential therapeutic effect of extracellular vesicles (EVs) that are derived from human liver stem cells (HLSCs) has been tested in an in vivo model of renal ischemia and reperfusion injury (IRI), that induce the development of chronic kidney disease (CKD). EVs were administered intravenously immediately after the IRI and three days later, then their effect was tested at different time points to evaluate how EV-treatment might interfere with fibrosis development. In IRI-mice that were sacrificed two months after the injury, EV- treatment decreased the development of interstitial fibrosis at the histological and molecular levels. Furthermore, the expression levels of pro-inflammatory genes and of epithelial-mesenchymal transition (EMT) genes were significantly reverted by EV-treatment. In IRI-mice that were sacrificed at early time points (two and three days after the injury), functional and histological analyses showed that EV-treatment induced an amelioration of the acute kidney injury (AKI) that was induced by IRI. Interestingly, at the molecular level, a reduction of pro-fibrotic and EMT-genes in sacrificed IRI-mice was observed at days two and three after the injury. These data indicate that in renal IRI, treatment with HLSC-derived EVs improves AKI and interferes with the development of subsequent CKD by modulating the genes that are involved in fibrosis and EMT.

Quinagolide Treatment Reduces Invasive and Angiogenic Properties of Endometrial Mesenchymal Stromal Cells.

Endometrial mesenchymal stromal cells (E-MSCs) extensively contribute to the establishment and progression of endometrial ectopic lesions through formation of the stromal vascular tissue, and support to its growth and vascularization. As E-MSCs lack oestrogen receptors, endometriosis eradication cannot be achieved by hormone-based pharmacological approaches. Quinagolide is a non-ergot-derived dopamine receptor 2 agonist reported to display therapeutic effects in in vivo models of endometriosis. In the present study, we isolated E-MSCs from eutopic endometrial tissue and from ovarian and peritoneal endometriotic lesions, and we tested the effect of quinagolide on their proliferation and matrix invasion ability. Moreover, the effect of quinagolide on E-MSC endothelial differentiation was assessed in an endothelial co-culture model of angiogenesis. E-MSC lines expressed dopamine receptor 2, with higher expression in ectopic than eutopic ones. Quinagolide inhibited the invasive properties of E-MSCs, but not their proliferation, and limited their endothelial differentiation. The abrogation of the observed effects by spiperone, a dopamine receptor antagonist, confirmed specific dopamine receptor activation. At variance, no involvement of VEGFR2 inhibition was observed. Moreover, dopamine receptor 2 activation led to downregulation of AKT and its phosphorylation. Of interest, several effects were more prominent on ectopic E-MSCs with respect to eutopic lines. Together with the reported effects on endometrial and endothelial cells, the observed inhibition of E-MSCs may increase the rationale for quinagolide in endometriosis treatment.

Extracellular vesicles from human liver stem cells inhibit renal cancer stem cell-derived tumor growth in vitro and in vivo.

Cancer stem cells (CSCs) are considered as responsible for initiation, maintenance and recurrence of solid tumors, thus representing the key for tumor eradication. The antitumor activity of extracellular vesicles (EVs) derived from different stem cell sources has been investigated with conflicting results. In our study, we evaluated, both in vitro and in vivo, the effect of EVs derived from human bone marrow mesenchymal stromal cells (MSCs) and from a population of human liver stem cells (HLSCs) of mesenchymal origin on renal CSCs. In vitro, both EV sources displayed pro-apoptotic, anti-proliferative and anti-invasive effects on renal CSCs, but not on differentiated tumor cells. Pre-treatment of renal CSCs with EVs, before subcutaneous injection in SCID mice, delayed tumor onset. We subsequently investigated the in vivo effect of MSC- and HLSC-EVs systemic administration on progression of CSC-generated renal tumors. Tumor bio-distribution analysis identified intravenous treatment as best route of administration. HLSC-EVs, but not MSC-EVs, significantly impaired subcutaneous tumor growth by reducing tumor vascularization and inducing tumor cell apoptosis. Moreover, intravenous treatment with HLSC-EVs improved metastasis-free survival. In EV treated tumor explants, we observed both the transfer and the induction of miR-145 and of miR-200 family members. In transfected CSCs, the same miRNAs affected cell growth, invasion and survival. In conclusion, our results showed a specific antitumor effect of HLSC-EVs on CSC-derived renal tumors in vivo, possibly ascribed to the transfer and induction of specific antitumor miRNAs. Our study provides further evidence for a possible clinical application of stem cell-EVs in tumor treatment.

Stem cell-derived extracellular vesicles inhibit and revert fibrosis progression in a mouse model of diabetic nephropathy.

Extracellular vesicles (EVs) that are derived from mesenchymal stromal cells (MSCs) have been shown to reprogram injured cells by activating regenerative processes. We herein investigate the potential therapeutic effect of EVs, shed by human bone marrow MSCs and by human liver stem-like cells (HLSCs), on the progression and reversion of fibrosis in a mouse model of diabetic nephropathy, as induced by streptozotocin. After the development of nephropathy, stem cell-derived EVs were administered weekly to diabetic mice for four weeks. The stem cell-derived EV treatment, but not the fibroblast EV treatment that was used as a control, significantly ameliorated functional parameters, such as albumin/creatinine excretion, plasma creatinine and blood urea nitrogen, which are altered in diabetic mice. Moreover, the renal fibrosis that develops during diabetic nephropathy progression was significantly inhibited in stem cell EV-treated animals. A correlation was found between the down regulation of several pro-fibrotic genes in renal tissues and the anti-fibrotic effect of HLSC and MSC EVs. A comparative analysis of HLSC and MSC EV miRNA content highlighted some common and some specific patterns of miRNAs that target predicted pro-fibrotic genes. In conclusion, stem cell-derived EVs inhibit fibrosis and prevent its progression in a model of diabetes-induced chronic kidney injury.

Human liver stem cell-derived extracellular vesicles enhance cancer stem cell sensitivity to tyrosine kinase inhibitors through Akt/mTOR/PTEN combined modulation.

It is well recognized that Cancer Stem Cells (CSCs) sustain the initiation, the maintenance and the recurrence of tumors. We previously reported that extracellular vesicles (EVs) derived from human liver stem cells (HLSCs) were able to limit tumor development. In this study, we evaluated whether EV derived from HLSCs could act in synergy with tyrosine kinase inhibitors (TKIs) on apoptosis of CSCs isolated from renal carcinomas. For this purpose, we administered to renal CSCs, HLSC-EVs and TKIs, as co-incubation or sequential administration. We found that HLSC-EVs in combination with Sunitinb or Sorafenib significantly increased renal CSCs apoptosis induced by low TKI dose. At variance, no synergistic effect was observed when bone marrow mesenchymal stem cell-derived EVs were used. In particular, renal CSCs chemosensitivity to TKIs was enhanced when HLSC-EVs were either co-administered with TKIs or added after, but not before. CSC apoptosis was also incremented at a percentage comparable to that of co-administration when TKIs were loaded in HLSC-EVs. By a mechanistic point of view, Akt/mTOR and Erk and Creb intracellular pathways, known to be pivotal in the induction of tumor growth and survival, appeared modulated as consequence of TKIs/HLSC-EVs co-administration. Together, our results indicate that the synergistic effect of HLSC-EVs with TKIs may increase the response to TKIs at low doses, providing a rational for their combined use in the treatment of renal carcinoma.

Role of HLA-G and extracellular vesicles in renal cancer stem cell-induced inhibition of dendritic cell differentiation.

BACKGROUND: Tumor immune-escape has been related to the ability of cancer cells to inhibit T cell activation and dendritic cell (DC) differentiation. We previously identified a tumor initiating population, expressing the mesenchymal marker CD105, which fulfills the criteria for definition as cancer stem cells (CD105(+) CSCs) able to release extracellular vesicles (EVs) that favor tumor progression and metastases. The aim of the present study was to compare the ability of renal CSCs and derived EVs to modulate the behavior of monocyte-derived DCs with a non-tumor initiating renal cancer cell population (CD105(-) TCs) and their EVs. METHODS: Maturation of monocyte-derived DCs was studied in presence of CD105(+) CSCs and CD105(-) TCs and their derived EVs. DC differentiation experiments were evaluated by cytofluorimetric analysis. T cell proliferation and ELISA assays were performed. Monocytes and T cells were purified from peripheral blood mononuclear cells obtained from healthy donors. RESULTS: The results obtained demonstrate that both CD105(+) CSCs and CD105(-) TCs impaired the differentiation process of DCs from monocytes. However, the immune-modulatory effect of CD105(+) CSCs was significantly greater than that of CD105(-) TCs. EVs derived from CD105(+) CSCs and in less extent, those derived from CD105(-) TCs retained the ability to impair monocyte maturation and T cell activation. The mechanism has been mainly related to the expression of HLA-G by tumor cells and to its release in a form associated to EVs. HLA-G blockade significantly reduced the inhibitory effect of EVs on DC differentiation. CONCLUSIONS: In conclusion, the results of the present study indicate that renal cancer cells and in particular CSCs and derived EVs impair maturation of DCs and T cell immune response by a mechanism involving HLA-G.