Figure 2.

MSC^IL-10 treatment during EVLP results in rapid dose-dependent hIL-10 elevation that decreases after lung transplantation. Pig double lungs subjected to 24 h cold ischemia were (A) connected to EVLP for 6 h, 20 × 10⁶ (n = 5) or 40 × 10⁶ (n = 6) MSCs^IL-10 were administered, and hIL-10 was measured using ELISA. (B) After MSC^IL-10 administration, EVLP perfusate hIL-10 levels were rapidly and dose-dependently elevated within minutes and continued to steadily increase for the duration of EVLP. (C) Lung tissue hIL-10 protein levels increased during EVLP, and (D) no significant differences were detected between samples taken from the upper or lower lobes, or from superficial or deep areas of the lung. (E) BAL hIL-10 was dose-dependently elevated 6 h after EVLP start. (F) After EVLP, the left lung was transplanted to a recipient pig that received methylprednisolone and cyclosporine A immunosuppression. (G) Recipient plasma hIL-10 protein levels peaked dose-dependently during the first 4 h after transplant reperfusion and declined to low levels within 1–2 days after transplantation. (H) Lung tissue hIL-10 protein levels were elevated 1 h after lung transplant reperfusion but were undetectable at 3 days. (I) Quantitative RT-PCR with human-specific primers of lung samples showed markedly increased hIL-10 mRNA expression at the end of EVLP, lower hIL-10 mRNA levels 1 h after transplant reperfusion and undetectable levels 3 days after transplantation. (J) Quantitative RT-PCR for human VEGF mRNA revealed low, but detectable levels of human VEGF mRNA in the lung 6 h after EVLP and 1 h after transplantation. (K) Lung tissue multiplex immunofluorescent and fluorescent in situ hybridization staining with human- (HLA Class I and ALU) and transgene-specific markers (FLAG tag; attached to the hIL-10 transgene) detected human MSCs^IL-10 in the pig lung 6 h after EVLP start. (K) MSCs^IL-10 co-expressed ALU, HLA Class I and FLAG, were relatively large, and (K,L) resided in duplicate or as (M,N) single cells. (L) Co-staining with endothelial (CD31) and epithelial (pan cytokeratin) markers indicated that MSCs^IL-10 mainly localized in the (K) lung interstitium and occasionally in the (L) alveolar space. (O) Quantitative PCR with ALU primers indicated the presence of human cells in the pig lungs at the end of EVLP, and 1 h after lung transplantation, but did not detect human genomic DNA in the transplant, or in any non-transplant recipient tissues 3 days after lung transplantation. Data mean ± standard deviation and analyzed by two-way ANOVA, or mixed-effect analysis in cases of missing data, using Šidák correction for multiple comparisons (B,C,E,G–J), or by one-way ANOVA with pairwise comparisons of the upper and lower lobes, or of superficial and deep location of MSC^IL-10 20 × 10⁶ and 40 × 10⁶ samples (D), or comparing other tissues to lung transplant (O). Results relative to GAPDH housekeeping gene (I,J). Quantitative PCR ng of human ALU DNA per 500 ng of total DNA, and * p < 0.0001 compared to all other tissues (O). Insets show magnified single-channel fluorescent images of the dashed area (K). BAL, bronchoalveolar lavage; EVLP, ex vivo lung perfusion; hIL-10, human interleukin-10; MSC, mesenchymal stromal cell; TX, transplantation.