Fig 4.

Hematopoietic stem cells receive microRNA loaded extracellular vesicles released by Cy5-RNA positive telocytes. (A) Experimental set-up: Cardiac telocytes are transfected with lipofectamine/Cy5-RNA complexes and release extracellular vesicles loaded with fluorescent-RNA into culture supernatant; hematopoietic stem cells incubated with supernatant receive the Cy5-RNA vesicles and become fluorescent; in control experiments, telocytes are not transfected with oligoRNA in absence of lipofectamine, the released vesicles does not have Cy5-RNA and the corresponding hematopoietic stem cells are negative for Cy5 fluorescence. (B) TC transfection with Cy5-RNA: Rat or mouse telocytes are positive for fluorescent Cy5 after 3–5 hrs incubation with a mixture of Cy5-RNA oligo and Lipofectamine RNAiMAX (red filled areas). Controls, non-fluorescent telocytes, represent cells incubated with oligoRNA in absence of lipids (dot plots). (C) Cy5-RNA positive hematopoietic stem/progenitor cells: Hematopoietic progenitors (LSK) are fluorescent after incubation with supernatant derived for Cy5-RNA transfected telocytes (sample), but not in case of supernatant derived from telocytes negative for Cy5 (control). LSK cells Lineage/7AAD negative, Sca1 positive and c-Kit positive. N = 4, P = 0.03 (D) Representative FACS plots of data presented in (C). Right plot: Hematopoietic stem cells (HSC) and their multipotent progeny (MPP), defined as cells LSK Flt3 negative, are positive for Cy5 when cultured in presence of medium conditioned by Cy5-RNA transfected telocytes. Left plot: in control experiments, HSC/MPP have been incubated with conditioned supernatant derived from non-fluorescent telocytes.