Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs

Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell–cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription–polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55–65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.

[1]  Hui Zhao,et al.  Elucidating the Secretion Proteome of Human Embryonic Stem Cell-derived Mesenchymal Stem Cells*S , 2007, Molecular & Cellular Proteomics.

[2]  A. Caplan,et al.  Mesenchymal stem cells as trophic mediators , 2006, Journal of cellular biochemistry.

[3]  J. Ingwall,et al.  Evidence supporting paracrine hypothesis for Akt‐modified mesenchymal stem cell‐mediated cardiac protection and functional improvement , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[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]  J. Freyssinet,et al.  Membrane microparticles: two sides of the coin. , 2005, Physiology.

[6]  C. Melief,et al.  B lymphocytes secrete antigen-presenting vesicles , 1996, The Journal of experimental medicine.

[7]  W. Filipowicz,et al.  Role of Dicer in posttranscriptional RNA silencing. , 2008, Current topics in microbiology and immunology.

[8]  J. Ingwall,et al.  Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells , 2005, Nature Medicine.

[9]  P. Fontana,et al.  Cell-derived microparticles in haemostasis and vascular medicine , 2009, Thrombosis and Haemostasis.

[10]  Cicek Gercel-Taylor,et al.  MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. , 2008, Gynecologic oncology.

[11]  B. Brenner,et al.  Microparticles bearing tissue factor and tissue factor pathway inhibitor in gestational vascular complications , 2009, Journal of thrombosis and haemostasis : JTH.

[12]  A. Simon,et al.  Endothelial microparticles in diseases , 2008, Cell and Tissue Research.

[13]  R. Rosell,et al.  Circulating MicroRNA Signatures of Tumor-Derived Exosomes for Early Diagnosis of Non-Small-Cell Lung Cancer. , 2009, Clinical lung cancer.

[14]  B. Brenner,et al.  Microparticles, thrombosis and cancer. , 2009, Best practice & research. Clinical haematology.

[15]  F. Miller Riding the waves: neural and nonneural origins for mesenchymal stem cells. , 2007, Cell stem cell.

[16]  M. Pittenger,et al.  Mesenchymal stem cells and their potential as cardiac therapeutics. , 2004, Circulation research.

[17]  Qian Liu,et al.  A high-throughput method to monitor the expression of microRNA precursors. , 2004, Nucleic acids research.

[18]  A. Pasquinelli,et al.  A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA , 2001, Science.

[19]  F. Slack,et al.  The let-7 family of microRNAs. , 2008, Trends in cell biology.

[20]  P. Doevendans,et al.  Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium. , 2008, Stem cell research.

[21]  Z. Mourelatos,et al.  A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. , 2005, Genes & development.

[22]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[23]  G. Hannon,et al.  C . elegans involved in developmental timing in Dicer functions in RNA interference and in synthesis of small RNA , 2001 .

[24]  B. Louis,et al.  Enjoy the Silence: The Story of let-7 MicroRNA and Cancer. , 2007, Current genomics.

[25]  W. Huttner,et al.  Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells , 2005, Journal of Cell Science.

[26]  Aled Clayton,et al.  Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids , 2006, Current protocols in cell biology.

[27]  M. Vilardell‐Tarrés,et al.  Circulating microparticles, lupus anticoagulant and recurrent miscarriages. , 2009, European journal of obstetrics, gynecology, and reproductive biology.

[28]  A. Shet Characterizing blood microparticles: Technical aspects and challenges , 2008, Vascular health and risk management.

[29]  N. Smalheiser Exosomal transfer of proteins and RNAs at synapses in the nervous system , 2007, Biology Direct.

[30]  J. Freyssinet,et al.  Pathophysiologic significance of procoagulant microvesicles in cancer disease and progression , 2009, Hämostaseologie.

[31]  Lianbo Yu,et al.  Detection of microRNA Expression in Human Peripheral Blood Microvesicles , 2008, PloS one.

[32]  M. Salto‐Tellez,et al.  Derivation of Clinically Compliant MSCs from CD105+, CD24− Differentiated Human ESCs , 2007, Stem cells.

[33]  H. Northoff,et al.  Cardioprotection and cardiac regeneration by mesenchymal stem cells. , 2008, Panminerva medica.

[34]  R. Shiekhattar,et al.  Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing , 2005, Cell.

[35]  J. Mattick,et al.  Small regulatory RNAs in mammals. , 2005, Human molecular genetics.