We previously reported the role of vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSCs) in protecting against neonatal hyperoxic lung injuries. Recently, the paracrine protective effect of MSCs was reported to be primarily mediated by extracellular vesicle (EV) secretion. However, the therapeutic efficacy of MSC-derived EVs and the role of the VEGF contained within EVs in neonatal hyperoxic lung injury have not been elucidated. The aim of the study was to determine whether MSC-derived EVs attenuate neonatal hyperoxic lung injury and, if so, whether this protection is mediated via the transfer of VEGF. We compared the therapeutic efficacy of MSCs, MSC-derived EVs with or without VEGF knockdown, and fibroblast-derived EVs in vitro with a rat lung epithelial cell line challenged with H₂O₂ and in vivo with newborn Sprague-Dawley rats exposed to hyperoxia (90%) for 14 days. MSCs (1 × 10⁵ cells) or EVs (20 µg) were administered intratracheally on postnatal day 5. The MSCs and MSC-derived EVs, but not the EVs derived from VEGF-knockdown MSCs or fibroblasts, attenuated the in vitro H₂O₂-induced L2 cell death and the in vivo hyperoxic lung injuries, such as impaired alveolarization and angiogenesis, increased cell death, and activated macrophages and proinflammatory cytokines. PKH67-stained EVs were internalized into vascular pericytes (22.7%), macrophages (21.3%), type 2 epithelial cells (19.5%), and fibroblasts (4.4%) but not into vascular endothelial cells. MSC-derived EVs are as effective as parental MSCs for attenuating neonatal hyperoxic lung injuries, and this protection was mediated primarily by the transfer of VEGF.

Background: Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response. Methods: Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation. Results: MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury. Conclusions: Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.

Though hypothermia is the only clinically available treatment for neonatal hypoxic-ischemic encephalopathy (HIE), it is not completely effective in severe cases. We hypothesized that combined treatment with hypothermia and transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) would synergistically attenuate severe HIE compared to stand-alone therapy. To induce hypoxia-ischemia (HI), male Sprague-Dawley rats were subjected to 8% oxygen for 120 min after unilateral carotid artery ligation on postnatal day (P) 7. After confirmation of severe HIE involving >50% of the ipsilateral hemisphere volume as determined by diffusion-weighted brain magnetic resonance imaging (MRI) within 2 h after HI, intraventricular MSC transplantation (1 × 105 cells) and/or hypothermia with target temperature at 32°C for 24 h were administered 6 h after induction of HI. Follow-up brain MRI at P12 and P42, sensorimotor function tests at P40–42, evaluation of cytokines in the cerebrospinal fluid (CSF) at P42, and histologic analysis of peri-infarct tissues at P42 were performed. Severe HI resulted in progressively increased brain infarction over time as assessed by serial MRI, increased number of cells positive for terminal deoxynucleotidyl transferase nick-end labeling, microgliosis and astrocytosis, increased CSF cytokine levels, and impaired function in behavioral tests such as rotarod and cylinder tests. All of the abnormalities observed in severe HIE showed greater improvement after combined treatment with hypothermia and MSC transplantation than with either therapy alone. Overall, these findings suggest that combined treatment with hypothermia and human UCB-derived MSC transplantation might be a novel therapeutic modality to improve the prognosis of severe HIE, an intractable disease that currently has no effective treatment.

Chang YS, et al. J Korean Med Sci. 2015 Oct;30(Suppl 1):S3-S11. https://doi.org/10.3346/jkms.2015.30.S1.S3

Jeong SY, et al. J Korean Med Sci. 2017 Oct;32(10):1717-1720. https://doi.org/10.3346/jkms.2017.32.10.1717

We recently showed that intraventricular transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) significantly attenuated posthemorrhagic hydrocephalus (PHH) and brain injury after severe intraventricular hemorrhage (IVH) in newborn rat pups. The purpose of this study was to optimize the timing of MSC transplantation for severe IVH. Severe IVH was induced by injecting 100 μl of blood into each ventricle of Sprague–Dawley rats on postnatal day 4 (P4). Human UCB-derived MSCs (1 × 10⁵cells in 10 μl of normal saline) were transplanted intraventricularly under stereotaxic guidance either early at P6 or late at P11. Serial brain MRIs and behavioral function tests, such as negative geotaxis and rotarod tests, were performed. At P32, brain tissue samples were obtained for histological and biochemical analyses. Intracerebroventricular transplantation of MSCs significantly attenuated the development of PHH, behavioral impairment, increased apoptosis and astrogliosis, reduced corpus callosum thickness and brain myelination, and upregulated inflammatory cytokines including interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) at P6 but not at P11 after induction of severe IVH. Intracerebroventricular transplantation of human UCB-derived MSCs attenuated PHH and brain injury after severe IVH in newborn rats in a time-dependent manner. Significant neuroprotection was only demonstrated when administered early at 2 days after induction but not late at 7 days after induction of severe IVH.