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Intrapleural delivery of mesenchymal stem cells: a novel potential treatment for pleural diseases - PubMed

Intrapleural delivery of mesenchymal stem cells: a novel potential treatment for pleural diseases

Zhao-hui Qin et al. Acta Pharmacol Sin. 2011 May.

Abstract

Aim: To develop a method to deliver mesenchymal stem cells (MSCs) into the pleural cavity for the treatment of pleural diseases.

Methods: MSCs were isolated from rat bone marrow of rats and labeled with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) or green fluorescent protein (GFP) using a lentiviral vector. Eighteen Sprague-Dawley (SD) rats were inoculated intrapleurally with 1×10(6) MSCs-DAPI. The distribution of the fluorescent cells was observed using fluorescent microscopy for the following 30 d. Another 12 rats inoculated intrapleurally with 1×10(6) MSCs-GFP were observed for 14 d.

Results: The isolated cells were typical MSC phenotypes and could differentiate into adipocytes, osteoblasts, and chondroblasts in vitro. Microscopic analysis revealed that the labeled cells adhered to the surface of the pleural cavity. The highest number of the labeled cells was found to be adhered to all specimens from the mediastinal pleura, but no labeled cells were detected in the lung parenchyma or other tissues/organs, such as the liver, kidney, spleen, and mesenterium. Incidentally, stomas were found in the mediastinal pleura. The recovered MSCs-GFP from the pleural cavity retained their ability to adhere and proliferate.

Conclusion: We have established a novel method for intrapleural delivery of MSCs. The distribution of intrapleurally delivered MSCs was found to be limited to the pleurae and the pleural cavity, thereby providing us with a new approach to further investigation of the therapeutic roles of MSCs in pleural diseases.

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Figures

Figure 1
Figure 1

Characterization of MSC phenotype. Flow cytometry of MSCs demonstrated that majority of MSCs were CD90 (A, 95.84%; B, 98.31%), CD29 (C, 99.7%) and CD73 (D, 89.5%) positive. In contrast, only a small portion of isolated cells were positive for hemopoietic cell surface makers, CD11b/c (A, 1.86%) and CD45 (B, 1.59%).

Figure 2
Figure 2

MSCs demonstrated multipotent property under standard in vitro differentiating condition. Differentiation into adipocytes (Oil Red staining, red) (A), osteoblasts (AKP staining, orange) (B) and (Alizarin staining, red) (C), and chondroblasts (Toluidin blue staining, blue) (D) was seen.

Figure 3
Figure 3

MSCs labeled with DAPI and GFP. MSCs stained with DAPI for 4 h, fluorescence microscopy (A), phase contrast (B) and merge (C). MSCs nuclei were stained and the fluorescent signal was high. MSCs harvested from above cells were cultured in vitro for 4 h (D, merge), 4 d (E, merge), and 10 d (F, merge, 2 d after passaged once). Relatively strong fluorescent signal was present in the cells even after 10 d post-labeling. Fluorescence microscopy of MSCs after transfection with pGC FU-GFP-Lentivirus for 7 d (G), 19 d (H, 9 d after passaged once), and 29 d (I, 2 d after passaged twice). (A–F), ×200; (G−I), ×100.

Figure 4
Figure 4

The MSCs-DAPI positive number of different tissues within two pleural cavities and pleural fixative solutions collected from total 16 experimental rats. For each bar, the maximum number was 16, since the tissue it represents was collected from 16 rats. While DAPI-labeled cells were found in all 64 mediastinal pleura specimens including the cardiac pericardium (100%) and in all 32 pleural fixative solutions (100%), they were found less frequently in other tissues like the epicardium (3/16=19%), the parietal pleura of both left and right chest wall (10/32=31%) and both left and right diaphragm (11/32=34%). In this figure, the column of mediastinal pleura represents only pleura located at both sides of right lung accessory lobe, while pericardium, which also belongs to the mediastinal pleura, was shown in a separate column. Right or Left, specimens collected from the right or left side of pleural cavities. bP<0.05, cP<0.01, compared with the mediastinal pleura including the pericardium.

Figure 5
Figure 5

Destination of MSCs-DAPI after being inoculated into right pleural cavity in healthy rats. All were merged images. Images from d 1: the right cardiac pericardium (A) and the surface of right lung (B). Images from d 4: the mediastinal pleura located at the right side of the right lung accessory lobe (C), the membranous part of right diaphragm (D), the left cardiac pericardium (E), and inner surface of the right chest wall (F). Images from d 14: the mediastinal pleura located at the right side of the right lung accessory lobe (G), and the surface of epicardium (H). Images from d 30: the mediastinal pleura located at the left side of the right lung accessory lobe (I), the surface of the left thymus (J), the pleural fixative solution from the left thorax (K), and the surface of kidney (L). Of all microphotographs, (B–E) and (G), ×100; (A), (F), (H), and (J–L), ×200; and (I), ×400. This figure showed that the cells can adhere only to the surfaces of two pleural cavities (ie left and right), including on the surface of the epicardium. MSCs adhered in the form of either cell mass of different shape or scattered cells, and found to be proned to adhere near small blood vessels (white arrows in A, C, D). No DAPI labeled cell was found on the surface of kidney.

Figure 6
Figure 6

Destination of MSCs-GFP after being inoculated into right pleural cavity in healthy rats. Images from d 1: the mediastinal pleura (A–F), the right thymus (G–I); d 4: the mediastinal pleura (J); d 7: the mediastinal pleura (K); d 14: the mediastinal pleura (L). “Halo” around MSCs-GFP aggregate was seen in image C (white arrows), indicating that interactions between MSCs and the pleural constituent cells occurred during adhesion. MSCs-GFP aggregate localized in a net of small blood vessels was shown in image (L). (A, D, G), fluorescence. (B, E, H), phase contrast. (C, F, I), merge. (J, K), fluorescence microscopy. (L), merge. (A–C, G–I, J, L), ×100; (K), ×200; (D–F), ×400.

Figure 7
Figure 7

Channels exist for MSCs and fluid to traverse between the right and left pleural cavities. Only a few seconds after methylene blue injection into the right side pleural cavity, blue fluid was found flowing into the left thorax (A), indicating that there were quick fluid channels that permitted fluid to pass through the mediastinum. Likewise, DAPI-labeled MSCs suspension was also injected into the right side pleural cavity, fluorescence cells were easily visualized in the fluid flowing into left thorax under fluorescence microscopy (B). Both images of DAPI-stained (C) and normal mediastinal pleura (D) demonstrated that there were many oval stomas of different sizes in the cell monolayer part of mediastinal pleura (white arrows), indicating that these may be the unidentified anatomical channels by which fluid and cells traverse between the two pleural cavities freely. (B–D), ×100.

Figure 8
Figure 8

MSCs-DAPI and MSCs-GFP recovered from the pleural cavity and cultured in vitro again. MSCs-DAPI recovered were cultured in vitro for 1 d after being inoculated for 14 d (A–C) and for 30 d (D–F). MSCs-GFP recovered were cultured in vitro for 3 d (G–H) and 17 d (J–L, 7 d after passaged once) after being inoculated for 1 d. Both recovered MSCs-DAPI and MSCs-GFP have the capability to adhere. However, only MSCs-GFP can proliferate. Beside MSCs-GFP, native cells from the pleural cavity also have property to adhere and proliferate (white arrows in I). (A, D, G, J), fluorescence microscopy. (B, E, H, K), phase contrast. (C, F, I, L), merge. (A–F), ×200; (G–L), ×400.

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