Biological Effects of Different Extracellular Vesicles Population on Reversal of Marrow Cells Radiation Damage

We have shown that Human or mouse mesenchymal stem cell (MSC) derived vesicles separated by differential centrifugation (300g, 10kg &100kg) can reverse radiation whole bone marrow (WBM) damage in vitro on the hematopoietic cell line FDC-P1 and in vivo on recovery of stem cells and differentiated blood cells after whole body irradiation. The vesicle separation is a classic method for preparations of exosomes. However, this separation isolates the smaller vesicles, discarding larger vesicles. Biologic healing effects of larger vesicles with or without smaller vesicles have not been investigated. Here, we investigated the healing effects of 3 different preparations of vesicles: 10k pellet (microvesicles), 100-10k pellet (exosomes) & 100k pellet (no 10k spin).

Three different fractions (10k pellet, 100-10k pellet &100k pellet) of murine/human MSC or murine WBM derived vesicles were isolated by differential centrifugation. FDC-P1 cells were exposed at 500cGy irradiation and cultured with or without the addition of 3 different preparations of murine WBM or human MSC-derived vesicles for 7 days. A significant increase in cell proliferation was observed after 3 different vesicle fractions treatments. However, exosomes were clearly inferior, microvesicles and the combined exosomes and microvesicles population were superior. The capacity of human MSC-derived vesicles of different fractions on reversal of murine bone marrow damage was also evaluated in vivo. Again, the combined fractions shown a significant increase donor chimerism in bone marrow at 6 month post-transplant with 5x's the level of engraftment compared to irradiation control. The 10k and 100-10k fractions showed intermediate healing. Exposure to vesicles in irradiated FDC-P1 cells downregulated phosphorylated H2AX, the DNA damage marker and decreased apoptosis marker, PARP cleavage part. We further investigated the effect of murine marrow MSC-derived vesicles on the gene expression in peripheral blood cells from radiation damaged mice. 28 genes were altered by irradiation, 22 showed partial or complete reversal of these alterations. The characterization of three different fractions of vesicles derived from murine bone marrow cells and human MSC were evaluated by miRNA realtime PCR array (total 750 miRNA). Several MSC-EV associated miRNA responsible for the reversal of radiation damage were evaluated. Our preliminary data shown that miR-221, miR654-3p and miR210-5p could partially reverse radiation damage in FDC-P1 cells

There are different biological effects in different extracellular vesicles populations. For reversal of radiation toxicity the most effective vesicle population would include both smaller (exosomes) and larger vesicles (microvesicles).

Acknowedgements: This work was support by the NIH grant 5UH2TR00080