Human Placental Mesenchymal Stem Cells and Derived Extracellular Vesicles Ameliorate Lung Injury in Acute Respiratory Distress Syndrome Murine Model

Abstract

This study investigates the therapeutic potential of human placental mesenchymal stem cells (P-MSCs) and their extracellular vesicles (EVs) in a murine model of acute respiratory distress syndrome (ARDS), a condition with growing relevance due to its association with severe COVID-19. We induced ARDS-like lung injury in mice using intranasal LPS instillation and evaluated histological changes, neutrophil accumulation via immunohistochemistry, bronchoalveolar lavage fluid cell count, total protein, and cytokine concentration, as well as lung gene expression changes at three time points: 24, 72, and 168 h. We found that both P-MSCs and EV treatments reduced the histological evidence of lung injury, decreased neutrophil infiltration, and improved alveolar barrier integrity. Analyses of cytokines and gene expression revealed that both treatments accelerated inflammation resolution in lung tissue. Biodistribution studies indicated negligible cell engraftment, suggesting that intraperitoneal P-MSC therapy functions mostly through soluble factors. Overall, both P-MSC and EV therapy ameliorated LPS-induced lung injury. Notably, at the tested dose, EV therapy was more effective than P-MSCs in reducing most aspects of lung injury.

Keywords: COVID-19; acute respiratory distress syndrome (ARDS); exosomes; extracellular vesicles (EVs); good manufacturing practice (GMP); immunomodulation; inflammation; mesenchymal stem cells (MSCs); preclinical model.

Figure 1

P-MSC characterization: (A) P-MSC morphology at passage 4. 80% confluence: elongated, fibroblastic-like, spindle-shaped cells. Original magnification ×100, phase-contrast light microscopy. The scale bar represents 100 µm. (B) Karyotyping analysis of P-MSCs at the second (left) and fourth passage (right) using G-banding. (C) Cell culture viability graph. (D) Population doubling time graph. (E) Cumulative population doubling time graph. *—p < 0.05 when compared with the earlier passage. P—passage; PDT—population doubling time. (F) Immunophenotypic characterization of cultured human placental stem cells.

Figure 2

EV characterization results. (A,B) Representative transmission electron microscopy micrographs of P-MSC EV; scale bar indicates 200 nm. (C) Nanoparticle tracking analysis of the total vesicles isolated from the P-MSC-conditioned medium. Line corresponding to average number and size of isolated particles, calculated from the mean of 3 videos lasting 60 s per sample.

Figure 3

Histological samples of lung sections from all groups (H&E stain) and semi-quantative grading of histological lung injury. Panels show representative images of histological sections. The scale bar indicates 100 µm. The graph displays the results of histological lung injury grading. Individual data points are shown. Significant p values are displayed as comparisons across groups at each time point. P-MSC—human placental stem cell-treated; EV—P-MSC-derived EV-treated. Columns represent median values, with 25th and 75th quartiles as error bars.

Figure 4

Myeloperoxidase immunohistochemistry samples of lung sections from all groups. The scale bar indicates 50 µm. Panels show representative images of immunohistochemical sections. Graphs display results from the quantitative analysis of MPO-positive cells in lung samples. Individual data points are shown (10 annotations per sample). Significant p values are displayed as comparisons across groups at each time point. P-MSC—human placental stem cell-treated; EV—P-MSC-derived EV-treated. Columns represent median values, with the 25th and 75th quartiles as error bars.

Figure 5

Total and relative cell counts of neutrophils, macrophages, and lymphocytes in bronchoalveolar lavage fluid (BALF). Individual data points are shown. Significant p values are displayed as comparisons across groups at each time point. P-MSC—human placental stem cell-treated; EV—P-MSC-derived EV-treated. Columns represent median values, with the 25th and 75th quartiles as error bars.

Figure 6

Bronchoalveolar lavage fluid total protein concentration and cytokine concentrations. Individual data points are shown. Significant p values are displayed as comparisons across groups at each time point. P-MSC—human placental stem cell-treated; EV—P-MSC-derived EV-treated. Columns represent median values, with the 25th and 75th quartiles as error bars.

Figure 7

Changes in Il1b, Tnf, Il17a, and Foxp3 gene expression in lung tissue. Gene expression changes are presented as fold changes in comparison with tissue from healthy mice. Individual data points are shown. Significant p values are displayed as comparisons across groups at each time point. P-MSC—human placental stem cell-treated; EV—P-MSC-derived EV-treated. Columns represent median values, with the 25th and 75th quartiles as error bars.