We have previously demonstrated that murine bone marrow cells co-cultured with lung opposite a cell-impermeable membrane express high levels of lung cell-specific genes (

Aliotta et al, Stem Cells, 2007;25(9):2245–56
). Greater changes in gene expression were noted in marrow cells co-cultured with radiation-injured lung. A factor smaller than the pore size of the cell-impermeable membrane (0.4μm) is responsible for these changes as cell-free conditioned media (CM) had a similar effect on co-cultured marrow cells. Lung-derived microvesicles were found to enter marrow cells in culture and may be among the factors responsible for these phenotypic changes. We wished to determine if these observations could be generalized to co-cultures using other murine organs and whether these changes in gene expression are tissue-specific. Whole bone marrow (WBM) cells were isolated from C57BL/6 mice and co-cultured with lung, liver, heart and WBM cells from C57BL/6 mice exposed to 1200 centigrey (cGy) of total body irradiation (TBI) or no radiation. Control WBM cells were co-cultured with no tissue (control). Co-cultured WBM cells were analyzed 7 or 14 days later by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). In addition, cell-free CM made from the same organs was co-cultured for 7 or 14 days with WBM cells which were then analyzed by RT-PCR. Alternatively, CM was analyzed for the presence of microvesicles by electron microscopy (EM) of ultracentrifuged (UCF) pelleted material. WBM co-cultured only with lung had increased gene expression of surfactant proteins A (Sp-A) and C (Sp-C, 89 and 334-fold increase vs. control, respectively) whereas WBM co-cultured only with brain had increased gene expression of Glial Fibrillary Acidic Protein (GFAP, 4.6-fold increase vs. control). Slight increases in Albumin expression were seen in all co-culture groups but expression was markedly elevated in WBM co-cultured with liver (162,657-fold increase vs. control). Expression of heart-specific markers, including Troponin I and T2, was seen in WBM co-cultured with heart but these levels were not significantly different from those of other co-culture groups. Radiation injury augmented expression of certain genes in co-cultured WBM, including Sp-A (1019 vs. 89-fold increase) in lung co-cultures and GFAP (24 vs. 4.6-fold increase) in brain co-cultures. WBM co-cultured with CM from all organs demonstrated similar changes in gene expression. In addition, pelleted material from UCF CM contained RNA that was specific to the tissue from which the CM was made. EM of UCF CM demonstrated numerous membranebound particles 50–200nm in size that were typical of microvesicles in appearance. These data suggest that changes seen in gene expression of co-cultured WBM are largely tissue-specific, depending on the tissue they are co-cultured with. Microvesicles released by various tissues in co-culture may be among the mediators responsible for the changes seen in WBM gene expression.

Topics:
albumins, chemical surfactants, coculture techniques, culture media, conditioned, gene expression, mice, pulmonary surfactants, reverse transcriptase polymerase chain reaction, rna, whole-body irradiation

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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