Abstract 15515: Exosomes from Adult Human CD34+ Stem Cells Transfer Crucial Pro-Angiogenic MiRNA Inducing Functional Changes in Target Cells
In early clinical trials, human CD34+ stem cells (CD34) have been shown to improve exercise tolerance in patients with myocardial ischemia and reduce amputation rates in limb ischemia. Pre-clinical studies suggest that the CD34 function via secreting paracrine factors, promoting neovascularization. Recently, we have shown that CD34 secrete exosomes (Exo) which comprise a major component of their paracrine pro-angiogenic effect. Exo are membrane bound nano-vesicles, which contain RNAs and protein. We hypothesized that CD34 Exo carry transcribable miRNA, which are transferred to the recipient cells to induce angiogenesis.
Methods and Results: We isolated RNA from two functionally distinct Exo: pro-angiogenic CD34 Exo purified from the adult human peripheral blood (PB) CD34 cell culture supernatants, and a control non-angiogenic Exo from PB total mononuclear cell (MNC) supernatants. Bioanalyzer analyses indicate that the CD34 Exo is enriched for small RNAs and miRNAs as compared to the CD34 cells themselves. The miRNAs were shown to be present inside the lumen of the Exo. Differential expression of miRNA between CD34 and MNC Exo was profiled using miRNA microarray. Known pro-angiogenic miRNA expression was measured by qRT-PCR. We detected higher expression of pro-angiogenic miRNAs in CD34 Exo as compared to MNC Exo- miR126 and miR 130a had ~ 18-fold and 10-fold higher expression in CD34 Exo. Interestingly, miRNA-126 in MNCs increased 4-fold after incubation with CD34 Exo, providing evidence of transfer. FACS analysis of CD34 Exo from cells transfected with Cy3 labeled miRNA confirms the release of Cy3-miRNA in CD34 Exo. Cy3-miRNA was detected in the intracellular punctate vesicles of recipient HUVECs by confocal microscopy when incubated with Cy3-miRNA-tagged CD34 Exo, demonstrating uptake. Effect of the transferred pro-angiogenic miRNAs on in vitro angiogenic activity and modulation of recipient cell gene expression is studied by loss of function-gain of function studies.
Conclusion: Our results demonstrate that adult human CD34+ cell derived exosomes carry and transfer pro-angiogenic miRNA to the recipient cells. It supports a mechanism of CD34+ cell communication involving intercellular traffic of miRNAs by exosomes to induce angiogenesis.
- © 2011 by American Heart Association, Inc.