Heparin Can Augment Extracellular Vesicle Yield by Inhibiting Reuptake in Donor Cells During Cell Propagation

Extracellular vesicles (EVs) mediate cell-to-cell communication in development, health and disease. Heparin is a common component in human induced pluripotent stem cell (hiPSC) media but its impact on hiPSC-EVs beyond stabilizing growth factors is unknown. Published evidence indicates heparin dose-dependent EV uptake inhibition in heterotypic target cells. We asked if heparin affects hiPSC-EV yield, storage, phenotype, or function. We used large-scale hiPSC-EV propagation to obtain pro-angiogenic hiPSC-EVs in presence or absence of escalating heparin doses and determined EV quantity, identity and functionality. Heparin delayed hiPSC colony-confluence without significantly affecting hiPSC viability, pluripotency markers and proliferation. Significantly elevated tetraspanin⁺/flotillin⁺ hiPSC-EV number (mean 367±31 EVs/cell/24 h at 200 IU heparin/mL) enabled efficient purification of > 10¹¹/mL functional EVs at superior particle/protein ratio > 10¹⁰/mg. Heparin dose-dependently inhibited EV uptake in hiPSC and endothelial progenitors, comparable to 5 IU/mL flavobacterium heparinase, and increased EV recovery after storage in NaCl but not in cell-conditioned media. EVs obtained from hiPSC at 1% O₂ with 200 IU/mL heparin maintained superior angiogenesis similarly to control hiPSC-EVs obtained without heparin. We conclude that heparin inhibits hiPSC-EV reuptake and stabilizes NaCl-cryopreserved EVs thus boosting EV recovery while maintaining pro-angiogenic function. Heparin presence and dose should therefore be documented particularly in PSC media.