Timing of umbilical cord blood derived mesenchymal stem cells transplantation determines therapeutic efficacy in the neonatal hyperoxic lung injury

Abstract

Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuates the hyperoxia-induced neonatal lung injury. The aim of this study was to optimize the timing of MSCs transplantation. Newborn Sprague-Dawley rats were randomly exposed to hyperoxia (90% for 2 weeks and 60% for 1 week) or normoxia after birth for 21 days. Human UCB-derived MSCs (5×10(5) cells) were delivered intratracheally early at postnatal day (P) 3 (HT3), late at P10 (HT10) or combined early+late at P3+10 (HT3+10). Hyperoxia-induced increase in mortality, TUNEL positive cells, ED1 positive alveolar macrophages, myeloperoxidase activity and collagen levels, retarded growth and reduced alveolarization as evidenced by increased mean linear intercept and mean alveolar volume were significantly better attenuated in both HT3 and HT3+10 than in HT10. Hyperoxia-induced up-regulation of both cytosolic and membrane p47(phox) indicative of oxidative stress, and increased inflammatory markers such as tumor necrosis factor-α, interleukin (IL) -1α, IL-1β, IL-6, and transforming growth factor-β measured by ELISA, and tissue inhibitor of metalloproteinase-1, CXCL7, RANTES, L-selectin and soluble intercellular adhesion molecule-1 measured by protein array were consistently more attenuated in both HT3 and HT3+10 than in HT10. Hyperoxia-induced decrease in hepatocyte growth factor and vascular endothelial growth factor was significantly up-regulated in both HT3 and HT3+10, but not in HT10. In summary, intratracheal transplantation of human UCB derived MSCs time-dependently attenuated hyperoxia-induced lung injury in neonatal rats, showing significant protection only in the early but not in the late phase of inflammation. There were no synergies with combined early+late MSCs transplantation.

Figure 1. Temporal profiles of inflammatory cytokines.

Tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6 levels measured with ELISA at P 1, 3, 5, 7, 10 and 14 in the rat lung tissue. NC, Normoxia control group; HC, hyperoxia control group. Data; mean±SEM. ^* P<0.05 compared to NC.

Figure 2. Survival curve.

Kaplan-Meier survival curve up to P 21 showing decreased survival. HC compared to NC, and improved survival in both HT3 and HT3+10, but not in HT10. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. ^* P<0.05 compared to NC.

Figure 3. Histology and morphometric analysis of the surviving P21 rat lung.

(A): Representative optical microscopy photomicrographs of the lungs stained with hematoxylin and eosin (scale bar = 100 µm). (B): Degree of alveolarization measured by the mean linear intercept (left) and mean alveolar volume (right). NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^* P<0.05 compared to NC, ^# P<0.05 compared to HC,^† P<0.05 compared to HT3, ^‡ P<0.05 compared to HT10.

Figure 4. TUNEL positive cells in the distal lungs of the P 21 rat pups.

(A): TUNEL positive cells were labeled with FITC (green) and the cell nuclei were labeled with propidium iodide (red) (Scale bar; 25 µm). (B): Number of observed TUNEL positive cells per high power field. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^* P<0.05 compared to NC, ^# P<0.05 compared to HC,^† P<0.05 compared to HT3, ^‡ P<0.05 compared to HT10.

Figure 5. Donor cell localization in the lung of the P 21 rats.

(A): Fluorescent microscopic observation of the PKH26 labeled human UCB-derived MSCs (donor cells, red) localized in the lungs of the P 21 newborn rats and the nuclei labeled with DAPI (blue) (Scale bar; 25 µm). (B): Number of PKH26 positive cells in the lung per high power field. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^† P<0.05 compared to HT3.

Figure 6. p47^phox, cytosolic subunit of nicotinamide adenine dinucleotide phosphate oxidase in the P21 rat lung tissue.

(A): Fluorescent microscopic observation of p47^phox (green) localized in the lungs of the P21 rats and the nuclei labeled with DAPI (blue) (Scale bar; 25 µm). (B): Representative western blots (top) and densitometric histograms (bottom) in the cytosol (left) and membrane (right) fractions of P21 rat lung homogenates. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^* P<0.05 compared to NC, ^# P<0.05 compared to HC.

Figure 7. Histograms of inflammatory cytokines and chemokines in the hyperoxic lung injury after MSCs transplantation.

TNF-α, IL-1α, IL-1β, IL-6, and TGF-β levels measured by ELISA (A) and TIMP-1, CXCL7, RANTES, L-selectin and sICAM-1 levels measured by protein array at P21 in the rat lung tissue (B,C). NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^* P<0.05 compared to NC, ^# P<0.05 compared to HC,^† P<0.05 compared to HT3, ^‡ P<0.05 compared to HT10.

Figure 8. Inflammation with fibrosis in the hyperoxic lung injury after MSCs transplantation.

ED1 positive cells indicative of alveolar macrophage were labeled with FITC (green) and the nuclei were labeled with DAPI (blue) (Scale bar; 25 µm) (at top) and number of observed ED1 positive cells per high power field (below) (A), myeloperoxidase activity (B), and collagen levels (C) of the rat P21 lung tissues. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^* P<0.05 compared to NC, ^# P<0.05 compared to HC,^† P<0.05 compared to HT3, ^‡ P<0.05 compared to HT10.

Figure 9. VEGF and HGF in the hyperoxic lung injury after MSCs transplantation.

Representative RT-PCR blots (at top) and densitometric histograms (below) for HGF (A) and VEGF measured with ELISA (B) in the P21 rat lungs. NC, Normoxia control group; HC, hyperoxia control group; HT3, hyperoxia with stem cell transplantation group at P3; HT10, hyperoxia with stem cell treatment group at P10; HT3+10, hyperoxia with stem cell treatment group at P3 and P10. Data; mean±SEM. ^* P<0.05 compared to NC, ^# P<0.05 compared to HC,^† P<0.05 compared to HT3, ^‡ P<0.05 compared to HT10.