Treatment of neonatal mouse hyperoxia-induced lung deficit with endothelial progenitor cells from bone marrow

Premature birth-related lung deficit caused by high oxygen treatment (hyperoxia) can result in chronic lung disease. Previous studies demonstrated that disruption of alveolarisation (septation of the lung) and pulmonary vascularisation can be observed after hyperoxia. Injection of cultured cells isolated from bone marrow with mesenchymal or endothelial progenitor phenotype can return lung morphology to normal after neonatal hyperoxia in mice. However, long-term efficiency, as well as any potential side effects of such cell therapies, remains under investigation. In this study we hypothesised that endothelial progenitor cells and plate-adherent cells can be isolated from bone marrow, and can be used for treating the effects of hyperoxia in the lungs of neonatal mice by inducing or supporting alveolarisation via promotion of vascularisation. The aim of this project was to compare a number of endothelial progenitor cell types, first in vitro, and then in vivo, to determine how injection of various cell types from mouse bone marrow affects the hyperoxia-treated and healthy lung, which would result in the optimisation of a new treatment model. Newborn mice were treated with 90% oxygen or left in room air for four days. Samples of tissues were collected from hyperoxia-treated and normoxia mice at 5, 28 and 56 days postpartum. It was discovered that alveolarisation remains affected until 56 days of age, but vascularisation recovers by 28 days of age. Suitable cell types were then obtained in order to treat the effects of hyperoxia. Cell sorting of bone marrow, in vitro differentiation and analysis for the presence of vessel-like structures revealed that the freshly-isolated Kdr-enriched cell fraction is effective in forming blood-vessel-like structures in vitro and might have in vivo potential. EphA3-enriched cells from different passages were also selected based on relatively rapid vessel-like structure formation in vitro, and unsorted plate-adherent cells from passage 0 were used as a control. Hyperoxia-treated and normoxia mice were then injected intraperitoneally with 1x104 cells (per 2 g of animal weight) of the different cell populations isolated from bone marrow at five days of age. Freshly-isolated cells sorted for Kdr, plate-adherent cells from bone marrow cultured for 7-10 days, and EphA3-enriched cells from passages 8, 12 and 13-15 were injected. PBS (saline) was injected as a control. After Kdr-enriched cells were injected, partial recovery from hyperoxia was observed at 28 days, for example, alveolar size was significantly smaller than in PBS-injected hyperoxia-treated mice. This was followed by full recovery at 56 days. However, when cells were injected in normoxia mice, alveolar size was significantly increased compared to control. After injection of cultured plate-adherent cells some abnormalities were observed, i.e. increased number of mature blood vessels. These cells did not assist in alveolarisation recovery after hyperoxia. After the injection of EphA3-enriched cells at passage 8, partial alveolar recovery was observed with some increase in alveolar size in normoxia mice, at passage 12 – no recovery was observed with some increase in alveolar size in normoxia mice, and at passages 13-15 – animal death and aberrant growth formations were observed. These results indicate that fresh Kdr-enriched cells (and potentially EphA3+ cells from passage 8) have a higher corrective potential compared to plate-adherent and long-term cultured cells in this model. Although, when these cells are applied in the absence of deficit, potential harmful effects might be caused.