Abstract
The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.
Highlights
Extracellular vesicles (EVs) are nanosized membrane-bound vesicles that may function as mediators of cell–cell communication by transfer of cellular proteins, lipids, and nucleic a cids[1]
Full-length blots are shown in Supplementary Figure S2A-F. (E) Tunable resistive pulse sensing on the conditioned medium from non-transfected M1 cells and stable transfected CD9truc-enhanced green fluorescent protein (EGFP) cells indicated that the size distribution of extracellular vesicles (EVs) is not affected the expression reporter proteins (n = 3). (F) Size-exclusion chromatography fractions of CD9truc-EGFP cell-conditioned medium showed full-length CD9truc-EGFP in fractions 1–3, while shedded EGFP was present in fractions 10–12 (n = 3)
While polyethylene glycol (PEG) precipitated cell-conditioned medium from M1 and M1-CD9truc-EGFP cells revealed enriched EV markers ALIX and flotillin, and not β-actin or nuclear Lamin A/C, EGFP was detected only in cell-conditioned medium from M1-CD9truc-EGFP cells (Fig. 1D, the full-length blots are shown in Supplemental Figure S2B-F)
Summary
Extracellular vesicles (EVs) are nanosized membrane-bound vesicles that may function as mediators of cell–cell communication by transfer of cellular proteins, lipids, and nucleic a cids[1]. The tetraspanins are considered valid markers of E Vs14 and have been widely used for the isolation and tracking of EVs. The tetraspanins have intracellular N- and C-terminals that enable genetic fusion of fluorescent reporter proteins and luciferase[15,16,17,18,19]. The protein concentration in each fraction is shown (G) Anti-GFP immunoprecipitation of cells conditioned medium co-isolates CD9truc-EGFP and EV markers ALIX and TSG101 only in M1 cells stable transfected with CD9truc-EGFP (n = 3). While N and C terminal fusion of reporter proteins to the tetraspanins enables tracking of cell-specific EVs, these reporter proteins do not allow for affinity isolation of the labeled EVs from biological fluids in that the tetraspanin terminals are located inside EVs12,13
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