Persistence of microvesicle-induced gene expression changes in murine marrow cells using an in vitro and in vivo model.

Abstract

e21086 Background: Microvesicles are subcellular entities that are derived from a variety of cells and contain DNA, RNA and protein. We have previously shown that microvesicles isolated from murine lung tissue (lung-derived microvesicles, LDMV) enter murine whole bone marrow cells (WBM) in culture and WBM that has consumed LDMV produce lung-specific mRNA and protein up to three weeks later. We wished to determine the persistence of these phenotypic changes in a long-term in vitro culture and an in vivo transplant model. Methods: In vivo study: cell-free conditioned media (CM) was made using murine lungs. WBM cells were then co-cultured with either LDMV, minced lung fragments (separated from WBM by a 0.4 micron, cell-impermeable membrane) or neither. One week later, cultured WBM was transplanted into cohorts of lethally-irradiated C57BL/6 mice. Recipient mice were sacrificed six weeks post transplant and WBM isolated from each recipient was lysed and examined for the presence of lung-specific genes, including surfactants A, B, C and D (Sp-A, B, C, D), aquaporin-5 (Aq-5), and clara cell specific protein (CCSP), via real-time PCR. In vitro study: CM was made in same fashion as with the in vivo study. One week later cells were washed and placed into a secondary culture, without lung. WBM was harvested at two week intervals and RNA and protein was extracted for real-time PCR and Western Blot analysis. Results: In vivo study: Mice transplanted with WBM exposed to LDMV express pulmonary epithelial cell genes in the cells of their bone marrow, liver, spleen and thymus 6 weeks after transplantation. In vitro study: WBM maintains expression of pulmonary epithelial cell genes and proteins in vitro up to 12 weeks after exposure to soluble factors released by lung cells. Conclusions: These findings suggest that LDMV-induced phenotypic alteration of marrow cells is not a transient phenomenon and that a lung cell-derived transcriptional agent is transferred to marrow cells in culture and responsible for these stable phenotypic changes.