Neonatal C57BL/6 mice (age, 24 h; weight, 12 g) were used in all experiments in the present study. The C57BL/6-green fluorescent protein (GFP) mice were purchased from the Animal Resource Center of the Fourth Military Medical University (Xi'an, China) and maintained in a temperature controlled environment (22–24°C) under a 12-h light/dark cycle with access to food and water ad libitum. The mice were randomized into various groups and weighed, and blood samples were collected. All animal procedures were approved by the animal ethics committee of Shandong University (Jinan, China) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996).

BMSCs were isolated from the bone marrow from the tibia and femurs of all four limbs of 6–8-week-old C57BL/6-GFP transgenic mice using a previously reported approach (22). Briefly, fresh BMSCs were isolated by flushing Dulbecco's modified Eagle's medium (DMEM; American Type Culture Collection, Manassas, MD, USA) containing 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) through the medullary cavity of the mouse femurs. The cells were isolated using a Ficoll density gradient centrifugation method (1.077; Sigma-Aldrich) at 800 x g for 20 min at room temperature. The isolated BMSCs were cultured (1×10⁶ cells per 100-mm cell culture dish (Corning Incorporated, Corning, NY, USA) and expanded in low glucose culture containing culture medium (GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal calf serum (Becton Dickinson; BD Biosciences, San Diego, CA, USA) at 37°C in 5% CO₂. After 48 h, the nonadherent cells were removed, fresh medium was added, and the culture was maintained for 7 days. The cells were then washed three times with Tris-buffered saline, which was followed by a 1-h incubation in the dark with fluorescein isothiocyanate, (FITC)-conjugated goat anti-rabbit IgG (H+L) secondary antibody (cat. no. ZF-0311; dilution, 1:200, ZSGB-Bio Co., Beijing, China) in phosphate-buffered saline (PBS). 4,6-Diamino-2-phenyl indole (DAPI) was used for nuclear counterstaining. Images of the cells were captured using a camera system connected to a fluorescence microscope (ECLIPSE 90i and NIS-Elements AR system, Nikon Corporation, Tokyo, Japan). The cells had characteristic immunoreactivities for cell markers CD44, CD117, CD34 and CD106 (Santa Cruz Biotechnology, Inc., CA, USA) on the basis of immunocytochemistry. Briefly, 1×10⁴ cells grown on a poly-l-lysine-coated 24-well plate were washed three times with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature, following which the cells were permeabilized with 0.1% Triton X-100/PBS for 20 min and blocked with 4% goat serum for 1 h. The cells were then incubated with rat monoclonal CD44 (1:100; cat. no. sc-18849; Santa Cruz Biotechnology, Inc.), CD106 (1:100; cat. no. ab24853; Abcam,Cambridge, MA, USA) and CD117 (1:100; cat. no. ab24870; Abcam) antibodies, and a goat polyclonal CD34 (1:100; cat. no. sc-7045, Santa Cruz Biotechnology, Inc.) antibody for 1 h at room temperature in the dark (23).

BMSCs were suspended with trypsin and 5×10⁵ cells, washed twice with PBS containing 0.5% bovine serum albumin (Sigma-Aldrich), were incubated with 1:200 dilutions of fluorochrome-conjugated specific or isotype control antibodies for 30 min at 4°C. The BMSCs were subsequently resuspended in PBS and incubated with specific or isotype control antibodies: Phycoerythrin (PE)/Cy5-conjugated anti-CD44 (cat. no. ab25579), FITC-conjugated anti-CD106 (cat. no. ab24853), PE-conjugated anti-CD34 (cat. no. ab23830), and FITC-conjugated anti-CD117 (cat. no. ab24870; all Abcam) resuspended in PBS and incubated with mouse anti-human monoclonal antibodies for 30 min at 4°C. After being washed, the cells were resuspended in PBS for fluorescence-activated cell sorting (FACS) analysis.

The neonatal C57BL/6 mice were randomly divided into the following four groups (n=10 in each group): Control, high oxygen, BMSCs alone and BMSCs/rHuEPO. Hyperoxia exposure of the neonatal mice was prepared, as previously reported (24). Briefly, the BPD group of neonatal C57BL/6 mice (age, 24 h) were placed in chambers in which the oxygen concentration was maintained at FiO₂=0.60. The exposure to hyperoxia was continuous, and the animals were exposed to the hypoxic environment for 14 days, with a brief interruption for animal care (<10 min/day). Subsequently, 1×10⁶ BMSCs were administered intravenously and 5,000 U/kg rHuEPO was administered by intraperitoneal injection, 1 h prior to and 7 days following hyperoxia exposure of the neonatal mice.

The lung tissues were prepared for histological examination, as previously described (24). Briefly, the mice were sacrificed by intra-peritoneal injections of pentobarbital (100 mg/kg) at 14 days old. Following sacrifice, the lungs were fixed with 4% paraformaldehyde solution overnight, and the left lower lobe was embedded in paraffin. Tissue sections were produced using a microtome set at 4–5 µm (Leica RM226; Leica Microsystems GmbH, Heidelberg Germany). Alveolarization was assessed by performing radial alveolar counts (RACs), according to standard methods, as previously described (25). Briefly, from the center of the respiratory bronchiole, a perpendicular line was drawn to the edge of the acinus, as defined by a connective tissue septum or the pleura, and the number of septa intersected by this line were counted. A total of five counts were performed for each animal, and the average count from the five high-power fields was randomly selected. These experiments were performed by two examiners blinded to treatment assignment.

On day 14, the mice were sacrificed by intracoronary perfusion of 10% KCl solution, and the lungs were removed and fixed in 4% paraformaldehyde/PBS for 24 h, followed by storage in 70% ethanol. The left lower lobe was embedded in paraffin, sectioned into 4–5 µm-thick sections, deparaffinized in xylene, and rehydrated by serial immersions in 100, 95, 85 and 75% ethanol, and 100% water. Following blocking with 4% normal goat serum in PBS, the slides were incubated with primary antibodies overnight at 4°C, and biotinylated HRP-conjugated rabbit IgG (H+L) polyclonal secondary antibody (1:200; cat. no. ZB-2306; ZSGB-Bio Co.) for 20 min at room temperature. The following primary antibodies were used: Rabbit polyclonal MMP-9 (1:200; cat. no. ab3889; Abcam) and VEGF (1:200; cat. no. ab46154; Abcam), which was followed by a 1-h incubation in the dark with tetrametrylrhodarnine isothiocyante-conjugated goat anti-rabbit IgG (H+L) secondary antibody (cat. no. ZF-0316; dilution, 1:200; ZSGB-Bio Co., Ltd). A total of 10 serial cortex and hippocampal sections (50 µm interval for each section) from each animal (n=10 for each group) were used to quantify each parameter. The staining was analyzed using the image-analyzing system, Image Pro Plus 6 (Media Cybernetics, Rockville, MD, USA), as previously described (26).

All data are expressed as the mean ± standard error of the mean. Comparisons of parameters between two groups were made using an unpaired Student's t-test. Comparisons of parameters among three groups were made using one-way analysis of variance, followed by Scheffe's multiple comparison test. Statistical analysis performed using the SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The surface markers of BMSCs were determined using FACS and showed that the BMSCs were positive for CD44 (93.14%) and CD106 (95.37%), but negative for CD34 (1.69%) and CD117 (2.58%; Fig. 1).

A total of 10 animals were represented in each treatment group and at each time point. The body weights of the animals were lower in the BPD group (positive control) when the mice were 7- (5.89±0.26 g) and 14- (7.95±0.22 g) days-old, compared with the mice exposed to room air (8.34±0.25 and 11.05±0.28, respectively, P<0.05). The body weights in the BMSC alone and BMSC/rHuEPO treatment groups were higher at 7 days old (6.84±0.20 and 7.42±0.23 g, respectively) and 14 days old (9.27±0.21 and 10.38±0.26 g, respectively), compared with the hyperoxia group (P<0.05). Weight preservation was most marked in the BMSCs/rHuEPO-treated mice (P<0.05; Fig. 2).

To investigate whether treatment with BMSCs and BMSCs/rHuEPO have beneficial effects on hyperoxia exposure-induced impairment in lung structure, morphometric analysis was performed using RACs. Compared with the room air control, RACs were lower in the 14-day-old C57BL/6 mice exposed to hyperoxia, but higher in the mice treated with BMSCs alone and with BMSCs/rHuEPO during hyperoxia on postnatal days 7 and 14, compared with those of the BPD group. In addition, improved weight preservation was shown in the BMSCs/rHuEPO-treated mice, compared with the mice treated with BMSCs alone (Fig. 3).

The results of the immunohistochemical staining analysis revealed that exposure of the neonatal mice to hyperoxia for 14 days resulted in significant increases in the protein expression levels of MMP-9 in the lungs, and a decrease in VEGF. The alterations in these proteins were significantly improved following treatment with BMSCs alone and with BMSCs/rHuEPO on day 14. Of note, the improvement in these protein expression levels were more marked in the BMSCs/rHuEPO group, compared with the BMSCs only group (Fig. 4).

On day 14 following the injection of BMSCs from the C57BL/6-GFP mice,cytochemical staining of vessel markers, PECAM (red) and VEGF (red), in the lung tissues were observed. Images were captured using a camera system connected to a fluorescence microscope (Fig. 5). However, the differentiation did not differ between the BMSCs alone and BMSCs/rHuEPO groups.