Human Endothelial Cell-Derived Extracellular Vesicles Mitigate Radiation-Induced Hematopoietic Injury

Endothelial cells (ECs) are the major participants in bone marrow microenvironment and key regulators of hematopoiesis. ECs regulate hematopoiesis at least in part by shedding the extracellular vehicles (EVs). We have previously demonstrated that mouse EC-derived EVs facilitate hematopoietic regeneration after ionizing irradiation (Int J Radiat Oncol Biol Phys. 2019,104(2):291-301).

To translate these mouse findings to the clinic, we evaluated if human sources of EC-derived EVs could be potential cellular therapy treatment option in case of nuclear disaster. We tested EVs derived from three different sources of ECs: human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (HMVEC), and primary cord blood endothelial progenitor cells (CB-EPCs). Human EVs from all three different sources of ECs were similar in size (nm) as measured by NanoSight (HUVEC: 177±51, HMVEC: 177.2±16.52, CB-EPC: 187.2±28.62) and positive for EC markers such as VEcadherin and CD31 as detected by RT-PCR. In addition, we demonstrated that these EVs had the capacity to facilitate irradiated (3 Gy) human CD34+ cell recovery using a 7-day hematopoietic stem cell culture system. Specifically, human EVs increased the numbers of total cells by 1.32 to 8.69 folds (HUVEC: 1.32±0.37, HMVEC: 5.26±0.95, CB-EPC: 8.69±3.41), total CD34+ cells by 1.91-4.28 folds (HMVEC: 4.28±1.63, CB-EPC: 1.91±0.51), and colony-forming cells by 1.68 to 2 folds (HUVEC: 1.68±0.31, HMVEC: 1.97±0.33, CB-EPC: 2±0.1).

Unlike HUVECs and HMVECs, which have limited cell expansion capacity, primary CB-EPCs are highly proliferative and still express EC markers and maintain their growth characteristics beyond 10+ passages, making them ideal candidate for use. Additionally, CB-EPCs promoted both in vitro and in vivo hematopoietic stem cell expansion as we showed in a recent publication (Int J Radiat Oncol Biol Phys. 2023 116(5):1163-117). We further evaluated the effects of CB-EPC derived EVs on hematopoiesis in vivo. NSG mice were first exposed to 2 Gy of total body irradiation and then 24 hours later injected with 5´10⁴ human CB CD34+ cells. The mice were then treated daily with EVs at a dose of 5´10⁹ or PBS for 7 days starting from day 1. Human cell engraftment was checked weekly by flow cytometry in peripheral blood. The results demonstrated that CB-EPC derived EVs significantly increased the percentage (0.2±0.4 vs.0.6±0.4, P=0.005) and absolute counts (1±1.5 vs. 2.27±1.23 cells/µl of blood, P=0.002) of human cells starting on day +21. These significant differences in the percentage (3.7±3.9 vs. 25.03±9.2, P=0.0003) and absolute counts (30.21±30.03 vs. 235±101.7 cells/µl of blood, P=0.0003) maintained at least until day +98. In addition, human CD19+ B cells in peripheral blood were also significantly increased in EV-treated group compared to the PBS control group starting on day +35 (0.10±0.13 vs. 1.34±0.8 cells/µl of blood, P=0.0003) and maintained at least until day +98 (26.31±28.25 vs. 208.6±94.29 cells/µl of blood, P=0.0003). We further measured hematopoietic stem/progenitor cell content in the bone marrow by flow cytometry on day 102. Significant increases of the percentages (0.8±0.2 vs. 1.39 ± 0.5, P=0.01) and absolute counts (120905±49028 vs. 228025±113195 cells/femur, P=0.01) of human CD34+CD38- cells were observed in EV-treated mice as compared to the PBS control mice.

Mechanistically, EVs were able to promote cell proliferation as measured by cell cycle analysis. EVs decreased the cells in G0 phase (55.28%±15.43% vs. 32.80%±4.6%, P=0.0007) and increased the cells in G2/S/M phase (12.40%±7.1% vs. 26.29%±3.89%, P=0.0001) compared to the medium control. The impact of EVs on cell cycle appeared to be regulated by cyclin-dependent kinases (CDKs) since EVs increased the expression of several CDKs including CDK1, CDK4, and CDK6.

In conclusion, our results have demonstrated that human EC-derived EVs can mitigate hematopoietic injury induced by irradiation. EVs are an appealing candidate for use as an off-shelf product as these EVs could be stored at -80 degree Celsius for up to 12 months without losing phenotypic and functional characteristics. Our findings indicate that EC-derived EVs are a promising therapeutic for enhancing hematopoietic recovery after radiation exposure.