Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity

Abstract

Rationale: Transplantation of human CD34(+) stem cells to ischemic tissues has been associated with reduced angina, improved exercise time, and reduced amputation rates in phase 2 clinical trials and has been shown to induce neovascularization in preclinical models. Previous studies have suggested that paracrine factors secreted by these proangiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34(+) cell transplantation.

Objective: Our objective was to investigate the mechanism of CD34(+) stem cell-induced proangiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved.

Methods and results: Exosomes collected from the conditioned media of mobilized human CD34(+) cells had the characteristic size (40 to 90 nm; determined by dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34(+) cell lineage marker protein, CD34. In vitro, CD34(+) exosomes replicated the angiogenic activity of CD34(+) cells by increasing endothelial cell viability, proliferation, and tube formation on Matrigel. In vivo, the CD34(+) exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34(+) cells but not from CD34(+) cell-depleted mononuclear cells had angiogenic activity.

Conclusions: Our data demonstrate that human CD34(+) cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34(+) exosomes may represent a significant component of the paracrine effect of progenitor cell transplantation for therapeutic angiogenesis.

Figure 1. Morphological analyses of exosomes from human CD34⁺ cells and MNCs

(a) Transmission electron micrograph of CD34⁺ cell (i) cytoplasm with MVBs enclosing numerous bilipidic layer-bound exosomes (Exo) (inset, arrows), (ii) inward invagination (arrows) in the MVB membrane indicate the beginning of exosome biogenesis, (iii) MVB fusing with cell membrane, (iv) Exosomes are secreted out from the cell (b) Electron microscopy of exosomes purified from CD34⁺ cell and MNC CM (c) Representative DLS number distribution measurement of isolated exosome population demonstrates a single peak (40–90 nm diameter) indicating they are free of contamination.

Figure 2. Biochemical analyses of exosomes from human CD34⁺cells and MNCs

Representative flow cytometry dot-plots showing detection of exosomal surface proteins (a) CD63 and (b) Annexin V bound to exposed phosphatidylserine, (c) Immunoblot showing exosomal luminal protein TSG101. (d) Flow cytometry dot-blot analysis for CD34 surface protein. The isolated exosomes were conjugated to 4-μm latex beads and stained for all flow cytometry analyses, Control represents non-specific antibody binding to the beads; the numbers inside the box represents the % of positive beads.

Figure 3. In-vitro assays

(a) HUVECs (2.5×10⁴) were treated with PBS, 2.0×10⁴ CD34⁺ cells, or with conditioned media (CM), exosomes (Exo), or exosome-depleted CM from 2.0×10⁴ CD34⁺ cells, and plated on Matrigel; tube length was measured 8 hours later and (b) expressed as percentage of saline-treated HUVECs; n= 6–9 (c) A representative dose-response of CD34⁺ exosome tube formation, evaluated in HUVECs incubated with exosomes from 1.5×10⁵ CD34⁺ cells and serially diluted with saline to the indicated ratios, (d) Viability (e) Proliferation of HUVECs (1×10⁴) in response to PBS, 2.5×10³ cells, or exosomes from 2.5×10³ cells, measured 20 hours later and expressed as percentage of PBS-treated HUVECs; n=3–6. *P<0.001 versus PBS, ^†P<0.05 versus Exo-depleted CM, ^‡P<0.05 versus MNCs or MNC exosomes.

Figure 4. In vivo assays

(a–b) Matrigel plug assay: Matrigel containing PBS, 5×10⁵ CD34⁺ cells, or exosomes from 5×10⁵ CD34⁺ cells was subcutaneously injected into mice, and the plugs were harvested 7 days later. (a) Sections from the plug were stained with isolectin to identify endothelial cells (brown) and vessel-like endothelial structures (arrows). (b) The plug was digested and CD31⁺ endothelial cells were quantified via flow cytometry; n=3–6. (c–d) Corneal angiogenesis assay: pellets containing PBS or exosomes from 1×10⁶ MNCs or CD34⁺ cells were implanted in the corneas of mice; corneas were harvested 7 days later, (c) stained with fluorescently labeled lectin identifying vascular structures, and (d) the extent of vessel growth was quantified; n=4. *P<0.05 versus PBS, ^‡P<0.01 versus MNC exosomes.