Lung-derived exosome uptake into and epigenetic modulation of marrow progenitor/stem and differentiated cells

Abstract

Background: Our group has previously demonstrated that murine whole bone marrow cells (WBM) that internalize lung-derived extracellular vesicles (LDEVs) in culture express pulmonary epithelial cell-specific genes for up to 12 weeks. In addition, the lungs of lethally irradiated mice transplanted with lung vesicle-modulated marrow have 5 times more WBM-derived type II pneumocytes compared to mice transplanted with unmanipulated WBM. These findings indicate that extracellular vesicle modification may be an important consideration in the development of marrow cell-based cellular therapies. Current studies were performed to determine the specific marrow cell types that LDEV stably modify.

Methods: Murine WBM-derived stem/progenitor cells (Lin-/Sca-1+) and differentiated erythroid cells (Ter119+), granulocytes (Gr-1+) and B cells (CD19+) were cultured with carboxyfluorescein N-succinimidyl ester (CFSE)-labelled LDEV. LDEV+ cells (CFSE+) and LDEV- cells (CFSE-) were separated by flow cytometry and visualized by fluorescence microscopy, analyzed by RT-PCR or placed into long-term secondary culture. In addition, murine Lin-/Sca-1+ cells were cultured with CFSE-labelled LDEV isolated from rats, and RT-PCR analysis was performed on LDEV+ and - cells using species-specific primers for surfactant (rat/mouse hybrid co-cultures).

Results: Stem/progenitor cells and all of the differentiated cell types studied internalized LDEV in culture, but heterogeneously. Expression of a panel of pulmonary epithelial cell genes was higher in LDEV+cells compared to LDEV - cells and elevated expression of these genes persisted in long-term culture. Rat/mouse hybrid co-cultures revealed only mouse-specific surfactant B and C expression in LDEV+ Lin-/Sca-1+cells after 4 weeks of culture, indicating stable de novo gene expression.

Conclusions: LDEV can be internalized by differentiated and more primitive cells residing in the bone marrow in culture and can induce stable de novo pulmonary epithelial cell gene expression in these cells for several weeks after internalization. The gene expression represents a transcriptional activation of the target marrow cells. These studies serve as the basis for determining marrow cell types that can be used for cell-based therapies for processes that injure the pulmonary epithelial surfaces.

Keywords: bone marrow cells; extracellular vesicles; pulmonary epithelial cells.

Fig. 1

Separation of LDEV+ and LDEV− bone marrow cells cultured with CFSE-labelled LDEV. (a) Whole bone marrow (WBM) cells (5.3×10⁴ cells/cm²) cultured without CFSE-labelled LDEV for 48 hours. (b) Lin-/Sca-1+, (c) CD19+, (d) Ter119+ and (e) Gr-1+ cells (all 5.3×10⁴ cells/cm²) cultured with CFSE-labelled LDEV (one mouse lung equivalent) for 48 hours. LDEV+ cells (R3) and LDEV− (**) could be detected and separated by flow cytometry. Y-axis, CFSE (FITC); X-axis, forward scatter (FSC).

Fig. 2

Flow cytometry–separated LDEV+ cells. (a) Lin-/Sca-1+, (b) CD19+, (c) Ter119+ and (d) Gr-1+ cells that have internalized CFSE-labelled LDEV. CFSE-labelled LDEV (FITC, green) can be found adjacent to the nucleus (DAPI, blue). Red bar=10 µm.

Fig. 3

Pulmonary epithelial cell mRNA transcripts detected in cells cultured with CFSE-labelled LDEV. (a) Cells cultured with CFSE-labelled LDEV (5.3×10⁴ cells/cm² with one mouse lung equivalent of LDEV for 48 hours) that are LDEV+ and (b) LDEV−. Gene expression is relative to cells not exposed to LDEV in culture (control) and is displayed as heat maps using a base 2 logarithmic scale. Cell culture experiments performed 3 times, and fold expression values are an average of all 3 experiments. *p>0.05 (Student's t-test), gene expression versus control cells.

Fig. 4

Gene expression profile of LDEV+ Gr-1+ and Lin-/Sca-1+ cells in long-term culture. Expression of pulmonary epithelial cell genes, stem/progenitor cell genes and granulocyte genes in (a) Gr-1+ cells and (b) Lin-/Sca-1+ cells that are LDEV+ relative to cells that are LDEV−. Cells were cultured for 2, 4, 6 or 8 weeks (5.3×10⁴ cells/cm²). Gene expression changes relative to LDEV− cells are displayed as heat maps using a base 2 logarithmic scale. Cell culture experiments performed 3 times, and fold expression values are an average of all 3 experiments. *p > 0.05 (Student's t-test), gene expression versus control cells.

Fig. 5

Sp-B and Sp-C expression in the mouse Lin-/Sca-1 cell/rat LDEV hybrid co-culture, short term. Expression (+ + +) or no expression (–) of mouse- or rat-specific Sp-B, Sp-C in mouse Lin-/Sca-1+ cells that were cultured for 48 hours with CFSE-labelled rat LDEV (5.3×10⁴ cells/cm² with 25 µg of rat LDEV). Shown is the presence or absence of expression in cells immediately after flow cytometry separation into LDEV+ cells and LDEV− cells (Day 0) and after 7 days of culture (Day 7).

Fig. 6

Sp-B and Sp-C expression in the mouse Lin-/Sca-1 cell/rat LDEV hybrid co-culture, long term. Mouse-specific (a) Sp-B and (b) Sp-C expression in LDEV+ (black bars) and LDEV− (white bars) cells from each passage (X-axis) after 4 weeks of culture. No rat-specific Sp-B or Sp-C expression was found in either cell population (not shown). Fold difference of gene expression is compared to cells cultured without LDEV (Y-axis).