Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects

Abstract

Previously we described a reliable method based on immunodepletion for isolating mesenchymal stem cells (MSCs) from murine bone marrow and showed that, after intracranial transplantation, the cells migrated throughout forebrain and cerebellum and adopted neural cell fates. Here we systemically administered MSCs purified by immunodepletion from male bleomycin (BLM)-resistant BALB/c mice into female BLM-sensitive C57BL/6 recipients and quantified engraftment levels in lung by real-time PCR. Male DNA accounted for 2.21 x 10(-5)% of the total lung DNA in control-treated mice but was increased 23-fold (P = 0.05) in animals exposed to BLM before MSC transplantation. Fluorescence in situ hybridization revealed that engrafted male cells were localized to areas of BLM-induced injury and exhibited an epithelium-like morphology. Moreover, purification of type II epithelial cells from the lungs of transplant recipients resulted in a 3-fold enrichment of male, donor-derived cells as compared with whole lung tissue. MSC administration immediately after exposure to BLM also significantly reduced the degree of BLM-induced inflammation and collagen deposition within lung tissue. Collectively, these studies demonstrate that murine MSCs home to lung in response to injury, adopt an epithelium-like phenotype, and reduce inflammation and collagen deposition in lung tissue of mice challenged with BLM.

Figures

Fig. 1.

Quantification of MSC engraftment in mouse lung by real-time PCR. (A) Relationship between threshold cycle number and the percentage of male genomic DNA within the indicated samples. The standard curve (blue dots) was generated by using samples containing from 100% to 0% male genomic DNA. (B) Histogram of the data in A showing the percentage of male genomic DNA within lung tissue or type II epithelial cells isolated from female mice 14 days after exposure to BLM, MSC administration (MSCs), or MSC administration after BLM exposure (BLM+MSC). Plotted values represent the arithmetic mean, and error bars represent the standard deviation (*,P = 0.05, and **, P = 0.001 by Student's ttest).

Fig. 2.

(A)A4-μm section of lung tissue from a male mouse hybridized with an FITC-conjugated Y chromosome paint probe and counterstained with ethidium bromide. The arrows indicate representative nuclei that contain the Y chromosome. (B) Localization of Y chromosome-positive nuclei (arrow) within lung tissue of a female mouse 14 days after BLM exposure and administration of male MSCs. (C and D) A series (–20) of images through the z axis were rendered from lung sections (as inB) to demonstrate that the probe hybridization signals (arrows) are associated specifically with cell nuclei.

Fig. 3.

Copurification of engrafted MSCs with type II epithelial cells. (A) Freshly isolated alveolar epithelial type II cells show darkly stained cytoplasmic granules after pap staining. (B) Cultured epithelial type II cells stain positively with an anticytokeratin antibody. (C) FISH analysis of cytospin preparations of alveolar type II cells (50,000 per slide) isolated from the lungs of a male mouse. (D) FISH analysis of alveolar epithelial type II cells isolated from female mice 14 days after exposure to BLM and administration of male MSCs. (Scale bars, 100 μM; magnification, ×40.)

Fig. 4.

Effect of BLM on osteopontin and MMP mRNA levels in lung. (A) Changes in osteopontin expression levels in lung tissue at various times after BLM exposure. The data points for C57BL/6 mice represent total RNA pooled from lung tissue of six animals. The data points for 129/J mice represent the mean of three to five separate expression values analyzing total RNA prepared from individual mice. (B) Total RNA was isolated from the lungs of untreated C57BL/6 mice (lane 1) or mice 14 days after exposure to BLM alone (lanes 2 and 3) or BLM exposure followed by MSC administration (lanes 4 and 5) and subjected to ribonuclease protection analysis as described inMaterials and Methods. Relative levels of each MMP transcript were determined by densitometry and normalized to the levels of GAPDH mRNA.

Fig. 5.

Effect of MSC engraftment on BLM-induced lung injury in mice. Low-magnification photomicrographs of tissue sections prepared from the lungs of a C57BL/6 mouse 14 days after saline exposure (A), 14 days after BLM exposure (B), and 14 days after BLM exposure and MSC administration (C). Note that the extent of BLM-induced inflammation, as evidenced by the wedge-shaped area of pneumonitis in B, is greatly reduced in C. TB, terminal bronchiole; PL, pleural surface. (Scale bar, 20 μm.)