Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing

Abstract

The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5+, Alix+, CD63+, and calnexin-). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.

Extracellular Vesicle-Derived microRNAs of Human Wharton’s Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis

“…To verify whether our procedures ensured the collection of a secretome with proangiogenic features comparable to that of a fetal dermal MSC secretome [ 31 , 32 ], and also to shed a light on the molecular mechanism underlying these features, we first performed a comparative Luminex-based quantification and confirmed the low amount of VEGF-A in the secretome of WJ-MSCs reported by others [ 25 , 26 , 27 ]. In particular, we found ~150 pg/mL VEGF-A in the WJ-MSC secretome vs. ~5000 pg/mL VEGF-A in the fetal dermal MSC secretome.…”

“…Our laboratory is dedicated to the isolation of MSCs from different tissues, including the human placenta [ 51 , 52 ], the fetal liver [ 53 ] and the fetal dermis [ 30 , 32 ], with the aim to elucidate the cell mechanism of action and potential challenges deriving from their use in cell-based therapies. Nevertheless, the shortage of human fetuses, whose availability largely depends on therapeutic abortions, encouraged us to consider additional sources of MSCs, such as the umbilical cord discarded with the placenta after birth.…”

“…The collection of secretome, including the measurement of soluble factors, was carried out as previously described [ 31 , 32 ]. Briefly, serum-free alpha-Minimum Essential Medium (MEM) (Gibco, Thermo Fisher Scientific) was added to 80% confluent cells, and collected 24 h later.…”

“…In vitro angiogenesis was conducted as previously described [ 31 , 32 ]. Briefly, 10,000 HUVECs (Life Technologies, Carlsbad, CA, USA) were suspended in the secretome of WJ-MSCs and plated onto matrigel from the in vitro angiogenesis assay kit (Millipore, Billerica, MA, USA).…”

“…Understanding the molecular mechanisms of the UC-MSC-mediated proangiogenic effect is necessary to improve the effectiveness of UC-MSC-based therapy. To gain insights into these mechanisms, we compared soluble components of Wharton’s jelly-(WJ)-MSCs with those of human fetal dermal MSCs [ 30 ]—these latter cells were previously characterized for the presence of proangiogenic soluble factors and EVs in their secretome [ 31 , 32 ]. Secretome from both cell types was subjected to a Luminex-based quantification to detect VEGF family members, such as VEGF-A, VEGF-D and placental growth factor (PlGF)-1 [ 33 , 34 ], and the chemokine stromal cell-derived factor (SDF)-1 alpha, which acts as proangiogenic stimulator in tissue repair [ 35 , 36 , 37 ].…”