Progenitor/stem cell fate determination: interactive dynamics of cell cycle and microvesicles.

We have shown that hematopoietic stem/progenitor cell phenotype and differentiative potential change throughout cell cycle. Lung-derived microvesicles (LDMVs) also change marrow cell phenotype by inducing them to express pulmonary epithelial cell-specific mRNA and protein. These changes are accentuated when microvesicles isolated from injured lung. We wish to determine if microvesicle-treated stem/progenitor cell phenotype is linked to cell cycle and to the injury status of the lung providing microvesicles. Lineage depleted, Sca-1+ (Lin-/Sca-1+) marrow isolated from mice were cultured with interleukin 3 (IL-3), IL-6, IL-11, and stem cell factor (cytokine-cultured cells), removed at hours zero (cell cycle phase G0/G1), 24 (late G1/early S), and 48 (late S/early G2/M), and cocultured with lung tissue, lung conditioned media (LCM), or LDMV from irradiated or nonirradiated mice. Alternatively, Lin-/Sca-1+ cells not exposed to exogenous cytokines were separated into G0/G1 and S/G2/M cell cycle phase populations by fluorescence-activated cell sorting (FACS) and used in coculture. Separately, LDMV from irradiated and nonirradiated mice were analyzed for the presence of adhesion proteins. Peak pulmonary epithelial cell-specific mRNA expression was seen in G0/G1 cytokine-cultured cells cocultured with irradiated lung and in late G1/early S cells cocultured with nonirradiated lung. The same pattern was seen in cytokine-cultured Lin-/Sca-1 cells cocultured with LCM and LDMV and when FACS-separated Lin-/Sca-1 cells unexposed to exogenous cytokines were used in coculture. Cells and LDMV expressed adhesion proteins whose levels differed based on cycle status (cells) or radiation injury (LDMV), suggesting a mechanism for microvesicle entry. These data demonstrate that microvesicle modification of progenitor/stem cells is influenced by cell cycle and the treatment of the originator lung tissue.

Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription.

OBJECTIVE: Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA. MATERIALS AND METHODS: Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-microm pore size), were analyzed using species-specific primers (for rat or mouse). RESULTS: These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena. CONCLUSION: These studies suggest that cellular systems are more phenotypically labile than previously considered.

Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation.

Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation-injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung-conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell-directed repair of injured tissue.