Mesenchymal Stem Cells Deliver Exogenous MicroRNA-let7c via Exosomes to Attenuate Renal Fibrosis.

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), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-β1, and TGF-β type 1 receptor (TGF-βR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that 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 coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-βR1 confirmed that miR-let7c attenuates TGF-β1-driven TGF-βR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is 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.

[1]  A. Vidal-Puig,et al.  Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity , 2011, PloS one.

[2]  N. Rajewsky,et al.  The evolution of gene regulation by transcription factors and microRNAs , 2007, Nature Reviews Genetics.

[3]  W. Stoorvogel Functional transfer of microRNA by exosomes. , 2012, Blood.

[4]  J. Lötvall,et al.  Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells , 2007, Nature Cell Biology.

[5]  Y. Kawahara Human diseases caused by germline and somatic abnormalities in microRNA and microRNA‐related genes , 2014, Congenital anomalies.

[6]  Wei Zhang,et al.  Immunosuppression effects of bone marrow mesenchymal stem cells on renal interstitial injury in rats with unilateral ureteral obstruction. , 2012, Cellular immunology.

[7]  R. Quigg,et al.  MicroRNA‐377 is up‐regulated and can lead to increased fibronectin production in diabetic nephropathy , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  K. Atkinson,et al.  Therapeutic applications of mesenchymal stromal cells. , 2007, Seminars in cell & developmental biology.

[9]  N. Perico,et al.  Autologous mesenchymal stromal cells and kidney transplantation: a pilot study of safety and clinical feasibility. , 2011, Clinical journal of the American Society of Nephrology : CJASN.

[10]  L. Moretta,et al.  Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. , 2008, Blood.

[11]  Lianghu Qu,et al.  The function of microRNAs in renal development and pathophysiology. , 2013, Journal of genetics and genomics = Yi chuan xue bao.

[12]  S. Gerson,et al.  Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH) , 2002, Bone Marrow Transplantation.

[13]  F. Sallustio,et al.  MicroRNAs in kidney diseases: new promising biomarkers for diagnosis and monitoring. , 2014, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[14]  W. Koh,et al.  Analysis of deep sequencing microRNA expression profile from human embryonic stem cells derived mesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of Hepatic Nuclear Factor 4 Alpha , 2010, BMC Genomics.

[15]  E. Brennan,et al.  Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFβR1. , 2013, Journal of the American Society of Nephrology : JASN.

[16]  H. Lan,et al.  TGF-β/Smad signaling in kidney disease. , 2012, Seminars in nephrology.

[17]  N. LaRusso,et al.  MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease. , 2008, The Journal of clinical investigation.

[18]  D. Kohn,et al.  Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors. , 1994, Blood.

[19]  A. Asawachaicharn,et al.  Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis , 2006, Experimental Neurology.

[20]  S. Ricardo,et al.  Combination therapy of mesenchymal stem cells and serelaxin effectively attenuates renal fibrosis in obstructive nephropathy , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[21]  Alan J Thompson,et al.  Autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: an open-label phase 2a proof-of-concept study , 2012, The Lancet Neurology.

[22]  C. Ricordi,et al.  Mesenchymal stromal (stem) cells to improve solid organ transplant outcome: lessons from the initial clinical trials , 2013, Current opinion in organ transplantation.

[23]  D. Hume,et al.  Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. , 2005, Journal of the American Society of Nephrology : JASN.

[24]  J. Bertram,et al.  Role of microRNAs in kidney homeostasis and disease. , 2012, Kidney international.

[25]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[26]  M. Rojas,et al.  Mesenchymal Stem Cells: A Promising Therapy for the Acute Respiratory Distress Syndrome , 2013, Respiration.

[27]  V. Patel,et al.  MicroRNAs and fibrosis , 2012, Current opinion in nephrology and hypertension.

[28]  N. Perico,et al.  Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation , 2013, Transplant international : official journal of the European Society for Organ Transplantation.

[29]  P. Delafontaine,et al.  Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. , 2009, Cell stem cell.

[30]  C. Bernard,et al.  Distinct Immunomodulatory and Migratory Mechanisms Underpin the Therapeutic Potential of Human Mesenchymal Stem Cells in Autoimmune Demyelination , 2013, Cell transplantation.

[31]  J. Kreidberg,et al.  MicroRNAs in renal development , 2013, Pediatric Nephrology.

[32]  John J Rossi,et al.  MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors , 2007, Proceedings of the National Academy of Sciences.

[33]  Fátima Sánchez-Cabo,et al.  Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells , 2011, Nature communications.

[34]  Merlin C. Thomas,et al.  Transforming growth factor-β1-mediated renal fibrosis is dependent on the regulation of transforming growth factor receptor 1 expression by let-7b. , 2014, Kidney international.

[35]  Tian Sheng Chen,et al.  Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs , 2009, Nucleic acids research.

[36]  S. Savitz,et al.  Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. , 2009, Stem cells and development.

[37]  V. Wheelock,et al.  Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s Disease , 2011, Molecular Neurobiology.

[38]  S. Messinger,et al.  Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. , 2012, JAMA.

[39]  S. Ricardo,et al.  Role of microRNA machinery in kidney fibrosis , 2014, Clinical and experimental pharmacology & physiology.

[40]  N. Perico,et al.  Human Bone Marrow Mesenchymal Stem Cells Accelerate Recovery of Acute Renal Injury and Prolong Survival in Mice , 2008, Stem cells.

[41]  Ohad Karnieli,et al.  Generation of Insulin‐Producing Cells from Human Bone Marrow Mesenchymal Stem Cells by Genetic Manipulation , 2007, Stem cells.

[42]  C. Lange,et al.  Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. , 2005, American journal of physiology. Renal physiology.

[43]  F. Slack,et al.  The let-7 microRNA represses cell proliferation pathways in human cells. , 2007, Cancer research.

[44]  R. Zhao,et al.  The Role of Chemokines in Mesenchymal Stem Cell Homing to Myocardium , 2011, Stem Cell Reviews and Reports.

[45]  S. Ricardo,et al.  Mesenchymal stem cells in kidney inflammation and repair , 2012, Nephrology.

[46]  Darwin J. Prockop,et al.  Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta , 1999, Nature Medicine.

[47]  P. Zamore,et al.  MicroRNA therapeutics , 2011, Gene Therapy.

[48]  Zhihong Wang,et al.  Cotransplantation of haploidentical hematopoietic and umbilical cord mesenchymal stem cells for severe aplastic anemia: successful engraftment and mild GVHD. , 2014, Stem cell research.

[49]  Oliver Eickelberg,et al.  Inhibition and role of let-7d in idiopathic pulmonary fibrosis. , 2010, American journal of respiratory and critical care medicine.

[50]  Timothy M. Williams,et al.  Human mesenchymal stem cells alter macrophage phenotype and promote regeneration via homing to the kidney following ischemia-reperfusion injury. , 2014, American journal of physiology. Renal physiology.

[51]  J. D. de Fijter,et al.  Autologous Bone Marrow‐Derived Mesenchymal Stromal Cells for the Treatment of Allograft Rejection After Renal Transplantation: Results of a Phase I Study , 2013, Stem cells translational medicine.

[52]  L. Muul,et al.  Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[53]  A. Hill,et al.  Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells , 2012, Nucleic acids research.

[54]  J. Mendell,et al.  MicroRNAs in cell proliferation, cell death, and tumorigenesis , 2006, British Journal of Cancer.

[55]  Yuhua Wang,et al.  A window onto siRNA delivery , 2013, Nature Biotechnology.

[56]  M. Wood,et al.  Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes , 2011, Nature Biotechnology.

[57]  S. Alexander,et al.  Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage , 2013, Laboratory Investigation.

[58]  Huanming Yang,et al.  Piglets cloned from induced pluripotent stem cells , 2012, Cell Research.

[59]  Qinxi Li,et al.  Axin determines cell fate by controlling the p53 activation threshold after DNA damage , 2009, Nature Cell Biology.

[60]  Frank P Barry,et al.  Mesenchymal stem cells avoid allogeneic rejection , 2005, Journal of Inflammation.

[61]  A. Xiang,et al.  Donor-Derived Mesenchymal Stem Cells Combined With Low-Dose Tacrolimus Prevent Acute Rejection After Renal Transplantation: A Clinical Pilot Study , 2013, Transplantation.

[62]  K. Pandit,et al.  MicroRNAs in idiopathic pulmonary fibrosis. , 2011, Translational research : the journal of laboratory and clinical medicine.