Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia

Abstract

Background: Human bone marrow-derived mesenchymal stem (stromal) cells (hMSCs) improve survival in mouse models of acute respiratory distress syndrome (ARDS) and reduce pulmonary oedema in a perfused human lung preparation injured with Escherichia coli bacteria. We hypothesised that clinical grade hMSCs would reduce the severity of acute lung injury (ALI) and would be safe in a sheep model of ARDS.

Methods: Adult sheep (30-40 kg) were surgically prepared. After 5 days of recovery, ALI was induced with cotton smoke insufflation, followed by instillation of live Pseudomonas aeruginosa (2.5×10(11) CFU) into both lungs under isoflurane anaesthesia. Following the injury, sheep were ventilated, resuscitated with lactated Ringer's solution and studied for 24 h. The sheep were randomly allocated to receive one of the following treatments intravenously over 1 h in one of the following groups: (1) control, PlasmaLyte A, n=8; (2) lower dose hMSCs, 5×10(6) hMSCs/kg, n=7; and (3) higher-dose hMSCs, 10×10(6) hMSCs/kg, n=4.

Results: By 24 h, the PaO2/FiO2 ratio was significantly improved in both hMSC treatment groups compared with the control group (control group: PaO2/FiO2 of 97±15 mm Hg; lower dose: 288±55 mm Hg (p=0.003); higher dose: 327±2 mm Hg (p=0.003)). The median lung water content was lower in the higher-dose hMSC-treated group compared with the control group (higher dose: 5.0 g wet/g dry [IQR 4.9-5.8] vs control: 6.7 g wet/g dry [IQR 6.4-7.5] (p=0.01)). The hMSCs had no adverse effects.

Conclusions: Human MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS.

Trail registration number: NCT01775774 for Phase 1. NCT02097641 for Phase 2.

Keywords: ARDS.

Figure 1

(A) Heart rate in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3).*p=0.01 in the higher-dose hMSC group vs the control group at 24 h by ANCOVA. There were no significant differences among the treatment groups by the GEE. Data are expressed as mean±SEM. (B) Systemic arterial blood pressure (MAP) (mean) in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). *p=0.004 in the higher-dose hMSC group vs the control group at 24 h by ANCOVA. ^$p<0.05 in the higher-dose hMSC group vs the control group during the 24 h period by the GEE. There was no significant difference between the lower-dose hMSC group and the control group by the GEE. Data are expressed as mean±SEM. (C) Systemic vascular resistance index (SVRI in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). No difference was found among the three groups at 24 h by ANCOVA or during the 24 h by the GEE. Data are expressed as mean±SEM. (D) Pulmonary capillary wedge pressure (PCWP) in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). ^*p=0.007 in lower-dose hMSC-treated sheep compared with control sheep by ANCOVA at 24 h. There were no significant differences among the treatment groups by the GEE. Data are expressed as mean±SEM.

Figure 2

(A) Pulmonary arterial pressure (PAP) in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). *p=0.008 in higher-dose hMSC-treated sheep compared with control sheep by ANCOVA at 24 h. ^#p<0.001 in lower-dose hMSC-treated sheep compared with control sheep by ANCOVA at 24 h. ^$p=0.04 in the higher-dose hMSC group vs the control group during the 24 h period by the GEE. There was no significant difference between the lower-dose hMSC group and the control group by the GEE. Data are expressed as mean±SEM. (B) Pulmonary vascular resistance index (PVRI) in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). No difference was found among the three groups at 24 h by ANCOVA. There were no significant differences among the treatment groups by the GEE. Data are expressed as mean±SEM.

Figure 3

(A) Peak airway pressure in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). There were no statistical differences in the lower-dose and higher-dose hMSC-treated sheep vs control sheep by ANCOVA at 24 h. There were no significant differences among the treatment groups by the GEE. Data are expressed as mean±SEM. (B) Pause airway pressure in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3). There were no differences in the lower and higher hMSC-treated groups vs the control group at 24 h by ANCOVA or during the 24 h experiment period by the GEE. Data are expressed as mean±SEM.

Figure 4

Total fluid balance (fluid in—urine output) in mL over the 24 h period in sheep treated with PlasmaLyte A alone (control) (n=8), lower-dose hMSCs (5×10⁶ cells/kg) (n=7) and higher-dose hMSCs (10×10⁶ cells/kg) (n=4). There were no differences among the three groups by Kruskal–Wallis equality-of-populations rank test. Data are expressed as median with 25–75% centiles.

Figure 5

The PaO₂/FiO₂ ratio (mmHg) in sheep treated with PlasmaLyte A alone (control) (T0: n=8; T24: n=6), lower-dose hMSCs (5×10⁶ cells/kg) (T0: n=7; T24: n=6) (n=7) and higher-dose hMSCs (10×10⁶ cells/kg) (T0: n=4; T24: n=3) (n=4). *p=0.003 in higher-dose hMSC-treated sheep compared with control sheep by ANCOVA at 24 h. ^#p=0.003 in lower-dose hMSC-treated sheep compared with control sheep by ANCOVA at 24 h. There were no significant differences among treatment groups over the 24 h period by the GEE. Data are expressed as mean±SEM.

Figure 6

Postmortem lung bloodless wet/dry weight ratio in sheep treated with PlasmaLyte A alone (control) (n=8), lower-dose hMSCs (5×10⁶ cells/kg) (n=7) and higher-dose hMSCs (10×10⁶ cells/kg) (n=4). *p=0.01 in higher-dose hMSC-treated sheep compared with control group by Mann–Whitney rank-sum test with Bonferroni adjustments. Data are expressed as median with 25–75% centiles.