Comparison of the therapeutic effects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury

Abstract

Introduction: Adipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI.

Methods: Human ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 × 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation.

Results: Both human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model.

Conclusions: Treatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS).

Figure 1

Persistence of hASCs and mASCs and change in body weight. (a) The percentages of total injected hASCs or mASCs present in the lung 20 hours after oropharyngeal administration for PBS-and LPS-challenged mice. Real-time RT-PCR analysis using human GADPH and eGFP primers was used to track and quantify the hASCs and mASCs, respectively. N = 4, 4, 4, and 5 for PBS + hASC, PBS + mASC, LPS + hASC, and LPS + mASC, respectively. (b) Changes in body weight following treatment with HBSS, hASC, or mASC were compared 24 hours after PBS or LPS challenge. N = 7, 6, 6, 16, 10, and 10 for Intact, PBS + hASC, PBS + mASC, LPS + HBSS, LPS + hASC, and LPS + mASC, respectively. Significance was defined as *** for P <0.001, for comparison of the experimental cell treatment groups to the HBSS-treated LPS-challenged mice. eGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hASCs, human adipose-derived stem cells; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 2

hASCs and mASCs improved severity of lung injury. Representative H&E lung sections 72 hours following LPS challenge for mice treated with (a) HBSS (N = 5), (b) hASCs (N = 5), or (c) mASCs (N = 5). Comparisons at the alveolar level (enlarged images) show decreased cellularity, septal thickening, and exudation when hASCs or mASCs were administered following LPS challenge. Levels of (d) protein and (e) albumin in the bronchoalveolar lavage fluid were used to assess the extent of vascular leakage after HBSS or ASC treatment. Control groups with intact or PBS-challenged mice were included to assess any changes in these levels due to the injection method or cell treatment. Values were presented as fold changes relative to intact, untreated mice. For (d) and (e), N = 3, 6, 6, 10, 9, and 10 for Intact, PBS + hASC, PBS + mASC, LPS + HBSS, LPS + hASC, and LPS + mASC, respectively. Significance was defined as ** and *** for P <0.01 and P <0.001, respectively, for comparison of the experimental cell treatment groups to the HBSS-treated LPS-challenged mice. ASC, adipose-derived stem cell; hASCs, human adipose-derived stem cells; HBSS, Hank's balanced salt solution; H&E, hematoxylin and eosin; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; PBS, phosphate-buffered saline.

Figure 3

Characterization of human and mouse ASC effects on inflammatory cell infiltration in the lung after LPS injury. Administration of hASCs or mASCs, when compared to HBSS, significantly decreased the (a) total cell number, (b) infiltrating neutrophil number as determined by a modified Wright-Giemsa stain, and (c) MPO activity in BALF 24 hours after LPS exposure. For (a) and (b), N = 3, 6, 6, 10, 9, and 10 for Intact, PBS + hASC, PBS + mASC, LPS + HBSS, LPS + hASC, and LPS + mASC, respectively. For (c), N = 4, 3, 5, 4, and 5 for PBS + hASC, PBS + mASC, LPS + HBSS, LPS + hASC, and LPS + mASC, respectively. Significance was defined as *, **, and *** for P <0.05, P <0.01, and P <0.001, respectively, as compared to the HBSS-treated LPS-challenged mice. ASC, adipose-derived stem cell; BALF, bronchoalveolar lavage fluid; hASCs, human adipose-derived stem cells; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; MPO, myeloperoxidase; PBS, phosphate-buffered saline.

Figure 4

Regulatory effects of hASCs and mASCs on inflammatory responses in the lung. The lung mRNA levels of various proinflammatory cytokines and chemokines 24 hours after PBS or LPS challenge were quantified using real-time RT-PCR. All levels were normalized to beta-actin and reported as fold changes compared to unchallenged and untreated mouse levels. Steady-state mRNA levels of (a) macrophage inflammatory protein 2 (MIP-2), (b) interleukin 1 alpha (IL-1α), (c) IL-1β, (d) MIP-1α, (e) tumor necrosis factor alpha (TNF-α), and (f) IL-10 are shown for PBS-or LPS-challenged mice injected with HBSS, hASCs, or mASCs. N = 2, 4, 4, 5, 4, and 5 for Intact, PBS + hASC, PBS + mASC, LPS + HBSS, LPS + hASC, and LPS + mASC, respectively. Significance was defined as *, **, and *** for P <0.05, P <0.01, and P <0.001, respectively, compared to control groups; significant differences between the LPS-challenged cell-treatment groups (LPS + hASC and LPS + mASC) were denoted by ## for P <0.01. hASCs, human adipose-derived stem cells; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 5

The effects of hASC and mASCs on inflammatory cytokine levels in the lung. Protein levels of various proinflammatory cytokines and chemokines 24 hours after PBS or LPS challenge were quantified for lung lysates using a multiplex immunoassay. All levels were normalized to total lung lysate protein concentration. Protein levels of (a) macrophage inflammatory protein 2 (MIP-2), (b) interleukin 1 alpha (IL-1α), (c) IL-1β, (d) interferon gamma (IFN-γ), (e) granulocyte macrophage colony-stimulating factor (GM-CSF), and (f) IL-10 are shown for PBS-or LPS-challenged mice injected with HBSS, hASCs, or mASCs. N = 4, 4, 5, 4, and 5 for PBS + hASC, PBS + mASC, LPS + HBSS, LPS + hASC, and LPS + mASC, respectively. Significance was defined as ** for P <0.01 compared to HBSS-treated LPS-challenged mice levels. hASCs, human adipose-derived stem cells; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; PBS, phosphate-buffered saline.