A functional corona around extracellular vesicles enhances angiogenesis during skin regeneration and signals in immune cells

Abstract

words)Allogeneic regenerative cell therapy has shown surprising results despite lack of engraftment of the transplanted cells. Their efficacy was so far considered to be mostly due to secreted trophic factors. We hypothesized that extracellular vesicles (EVs) can also contribute to their mode of action. Here we provide evidence that EVs derived from therapeutic placental-expanded (PLX) stromal cells are potent inducers of angiogenesis and modulate immune cell proliferation in a dose-dependent manner.Crude EVs were enriched >100-fold from large volume PLX conditioned media via tangential flow filtration (TFF) as determined by tunable resistive pulse sensing (TRPS).Additional TFF purification was devised to separate EVs from cell-secreted soluble factors. EV identity was confirmed by western blot, calcein-based flow cytometry and electron microscopy. Surface marker profiling of tetraspanin-positive EVs identified expression of cell-and matrix-interacting adhesion molecules. Differential tandem mass tag proteomics comparing PLX-EVs to PLX-derived soluble factors revealed significant differential enrichment of 258 proteins in purified PLX-EVs involved in angiogenesis, cell movement and immune system signaling. At the functional level, PLX-EVs and cells inhibited T cell mitogenesis. PLX-EVs and soluble factors displayed dose-dependent proangiogenic potential by enhancing tube-like structure formation in vitro.Our findings indicate that the mode of PLX action involves an EV-mediated proangiogenic function and immune response modulation that may help explaining clinical efficacy beyond presence of the transplanted allogeneic cells.

Self-assembly of differentiated progenitor cells facilitates spheroid human skin organoid formation and planar skin regeneration

“…Purified hiPSC-ECs formed pronounced vascular networks in the presence but not absence of hPL. To gain further insight into the mechanism of hPL-based angiogenesis support 26 , 30 we purified extracellular vesicles (EVs) from hPL, because we observed in another study that EVs can mediate such trophic effects 48 . The hPL-derived presumably platelet lipoprotein-enriched EVs 48 supported angiogenesis of hiPSC-ECs significantly in a dose-dependent manner even in the absence of otherwise essential externally added vascular endothelial growth factor (VEGF).…”

“…The clonogenic potential of fibroblasts, endothelial cells and hiPSC-derived cells was assessed according to published protocols 44 , 47 . The purification of extracellular vesicles from hPL supporting the angiogenic potential of ECs and hiPSC-ECs as well as the network formation assay was previously established and performed according to a published protocol 48 .…”

Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles

“…Particle-rich media, due to supplementation with human platelet lysate or fetal bovine serum, were instantly used to acquire bone marrow stromal cell-derived EVs for cell-free treatment of graft-versus-host disease [ 31 ]. Alternatively, pre-existing particles could be depleted before initiation of cell culture [ 32 ]. Chemically defined serum-free medium offers the advantage of a lower initial particle count, thus enabling a more precise quantification of EVs secreted by donor cells into the conditioned medium over time.…”

Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

“…Purified hiPSC-ECs formed pronounced vascular networks in the presence but not absence of hPL. To gain further insight into the mechanism of hPL-based angiogenesis support 11,13 we purified extracellular vesicles (EVs) from hPL, because recent evidence indicated that EVs can mediate such trophic effects 41 . The hPL-derived EVs supported angiogenesis of hiPSC-ECs significantly in a dose-dependent manner even in the absence of otherwise essential externally added vascular endothelial growth factor (VEGF).…”

“…The clonogenic potential of fibroblasts, endothelial cells and hiPSC-derived cells was assessed according to published protocols 50,51 . The purification of extracellular vesicles from hPL supporting the angiogenic potential of ECs and hiPSC-ECs as well as the network formation assay was previously established and performed according to a published protocol 41 .…”