Stable cell fate changes in marrow cells induced by lung-derived microvesicles

Abstract

Background: Interest has been generated in the capacity of cellular-derived microvesicles to alter the fate of different target cells. Lung, liver, heart and brain-derived vesicles can alter the genetic phenotype of murine marrow cells; however, the stability of such changes and the mechanism of these changes remain unclear. In the present work, we show that lung-derived microvesicles (LDMV) alter the transcriptome and proteome of target marrow cells initially by mRNA and regulator(s) of transcription transfer, but that long term phenotype change is due solely to transfer of a transcriptional regulator with target cell.

In vivo studies: Whole bone marrow cells (WBM) were co-cultured with LDMV (both isolated from male C57BL/6 mice) or cultured alone (control). One week later, cultured WBM was transplanted into lethally-irradiated female C57BL/6 mice. Recipient mice were sacrificed 6 weeks later and WBM, spleens and livers were examined for the presence of lung-specific gene expression, including surfactants A, B, C and D, aquaporin-5, and clara cell specific protein, via real-time RT-PCR. Immunohistochemistry was also performed on lungs to determine the number of transplanted marrow-derived (Y chromosome+) type II pneumocytes (prosurfactant C+). Mice transplanted with LDMV co-cultured WBM expressed pulmonary epithelial cell genes in the cells of their bone marrow, livers and spleens and over fivefold more transplanted marrow-derived Y+/prosurfactant C+cells could be found in their lungs (vs. control mice).

In vitro studies: WBM (from mice or rats) was cultured with or without LDMV (from mice or rats) for 1 week then washed and cultured alone. WBM was harvested at 2-week intervals for real-time RT-PCR analysis, using species-specific surfactant primers, and for Western Blot analysis. Proteomic and microRNA microarray analyses were also performed on cells. LDMV co-cultured WBM maintained expression of pulmonary epithelial cell genes and proteins for up to 12 weeks in culture. Surfactant produced at later time points was specific only to the species of the marrow cell in culture indicating de novo mRNA transcription. These findings, in addition to the altered protein and microRNA profiles of LDMV co-cultured WBM, support a stable transcriptional mechanism for these changes.

Conclusions: These data indicate that microvesicle alteration of cell fate is robust and long-term and represents an important new aspect of cellular biology.

Keywords: bone marrow cells; bone marrow transplant; lung; microvesicles; transcription factor.

Fig. 1

Pulmonary epithelial cell gene expression in marrow cells co-cultured with LDMV. Expression of (A) Sp-A, (B) Sp-B, (C) Sp-C, (D) Sp-D, (E) CCSP, (F) Aq-5 in WBM cells at various time points after an initial co-culture period of 1 week with LDMV. Data expressed as a fold difference compared with WBM cultured without LDMV for an equivalent period of time. *≤0.01, wilcoxon rank sum, 3 experiments, n=5–8, each time point.

Fig. 2

Pulmonary epithelial cell protein expression in marrow cells co-cultured with LDMV. (A) Prosurfactant B-expressing WBM cell (solid arrow) 12 weeks after exposure to LDMV in co-culture. (B) Prosurfactant B-expressing murine lung cell (dashed arrows). (C) WBM cells cultured without LDMV do not express prosurfactant B. (A–C) DAPI and Texas Red filters. (D) Western blot of WBM cells 12 weeks after exposure to LDMV in co-culture (lanes 1–4) and lung cells (+) express prosurfactant B whereas WBM cells cultured without LDMV (−) do not express prosurfactant B. Representative data from 1 of 3 experiments are shown.

Fig. 3

Rat/mouse hybrid co-culture, WBM and lung. Mouse-specific Sp-B (solid blue line) and Sp-C (dashed blue line), rat-specific Sp-B (sold red line) and Sp-C (dashed red line) expression in (A) murine WBM co-cultured with rat-derived lung and (B) rat-derived WBM co-cultured with murine lung. Data expressed as a fold difference compared with murine or rat-derived WBM cultured without lung for an equivalent period of time. *≤0.01, wilcoxon rank sum, 3 experiments, n=6–9, each time point.

Fig. 4

Rat/mouse hybrid co-culture, WBM and liver. Mouse-specific ABCA1 (solid blue line) and albumin (dashed blue line), rat-specific ABCA1 (sold red line) and albumin (dashed red line) expression in (A) murine WBM co-cultured with rat-derived liver and (B) rat-derived WBM co-cultured with murine liver. Data expressed as a fold difference compared with murine or rat-derived WBM cultured without liver for an equivalent period of time. *≤0.01, wilcoxon rank sum, 2 experiments, n=6, each time point.

Fig. 5

Transplanted marrow-derived type II pneumocytes. (A) Prosurfactant C+cell (white arrow, Texas Red filter), that is also (B) Y chromosome+(FITC filter) and (C) nucleated (DAPI filter) representing a (D) transplanted bone marrow cell-derived type II pneumocyte (merge) in the lungs of lethally irradiated female mice 6 weeks after transplantation with male murine WBM co-cultured with LDMV (63×magnification, room temperature). (E) Y+/prosurfactant C+cells, expressed as a percentage of all DAPI+lung cells, in mice transplanted with WBM co-cultured with LDMV, WBM cultured alone or uncultured WBM. *≤0.01, wilcoxon rank sum, 1 experiments, n=6–12.

Fig. 6

Altered proteome of Lin-cells co-cultured with LDMV. Relative expression of proteins in Lin– cells co-cultured with LDMV compared to Lin– cells cultured in the absence of LDMV. Up regulated proteins in LDMV co-cultured Lin– cells (34) are proteins common to both cell populations but more “Heavy”-labeled forms of these proteins were identified relative to “Light”-labeled forms. Down reregulated proteins (18) are proteins common to both cell populations but fewer “Heavy”-labeled forms of these proteins were identified relative to “Light”-labeled forms.

Fig. 7

Altered microRNA profile of Lin-cells co-cultured with LDMV. Relative expression of microRNA in Lin– cells co-cultured with LDMV compared to Lin– cells cultured in the absence of LDMV. 159 species common to both cell populations are up regulated in LDMV co-cultured Lin– cells and 101 species common to both cell populations are down regulated in LDMV co-cultured Lin– cells.

Fig. 8

Summary of findings, in vitro and in vivo persistence assays. Lung cells shed microvesicle-based transcriptional regulators which are internalized by cells of the bone marrow. Changes in microvesicle-modified cells are persistent in vitro, as these cells continue to express pulmonary epithelial cell mRNA and protein up to 12 weeks later, and in vivo after transplantation into lethally-irradiated mice, as they lead to the expression of pulmonary epithelial cell mRNA in non-pulmonary tissue and preferentially engraft the lung as functioning type II pneumocytes.