The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), researchers at Monash University report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c, selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared to non-targeting control (NTC)-MSCs. In vitro analysis confirmed the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-β1 was repressed following the addition of isolated exosomes or indirect co-culture of miR-let7c-MSCs, compared to NTC-MSCs. Furthermore, the co-transfection of NRK52E cells using the 3’UTR of TGF-βR1 confirmed that miR-let7c attenuates TGF-β1-driven TGF-βR1 gene expression.
(a) Flow cytometry and fluorescence microscopy for GFP+ expression showed the transfection efficiency of MSCs with miR-let7c, that were karyotypically normal. (b) Successful transduction of MSCs with miR-NTC was demonstrated by fluorescence microscopy and flow cytometry. The transfected cells also displayed karyotypically normality. (c) MSC displayed multilineage differentiation potential in vitro, differentiating into osteocytes, evidenced by osteocalcin staining (magnification x200), adipocytes indicated by the presence of lipid droplets stained with Oil Red-O (magnification x400) and chondrocytes shown by the presence of aggrecan staining (magnification x200). (d) In addition to displaying a normal morphology, the transduced MSCs, miR-let7c-MSC displayed a normal proliferative ability. Abbreviations: GFP, green fluorescent protein, NTC, non-targeting control; MSCs, mesenchymal stem cells. Scale bars: Panel B = 100µm.
Taken together, the effective anti-fibrotic function of engineered MSCs are able to selectively transfer miR-let7c to damaged kidney cells, and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.