This study was approved by the Institutional Review Board of Samsung Medical Center and by Medipost, Co., Ltd., Seoul, Korea. UCB was collected from umbilical veins after neonatal delivery with informed consent from pregnant mothers. As previously reported, mesenchymal stem cells were isolated from human UCB, and cultivated.5,6 The cells expressed CD105 (99.6%) and CD73 (96.3%), but not CD34 (0.1%), CD45 (0.2%) or CD14 (0.1%), and they were positive for human leukocyte antigen (HLA)-AB (96.8%) but not HLA-DR (0.1%).5 They expressed pluripotency markers, such as octamer bindings transcription factor 4 (30.5%)7 and stage specific embryonic antigen 4 (67.7%).8 Human UCB-derived MSCs differentiated into various cell types, including respiratory epithelium, osteoblasts, chondrocytes and adipocytes, in response to specific induction stimuli.5,6 We have confirmed the differentiation potential and karyotypic stability of the human UCB-derived MSCs up to the 11th passage.5,6

The Animal Care and Use Committee of Samsung Biomedical Research Institute, Seoul, Korea reviewed and approved the described experimental protocol. This study was also done in accordance with the Institutional and National Institutes of Health guidelines for laboratory animal care. Timed pregnant Sprague Dawley rats (Orient Co., Seoul, Korea) were housed in individual cages with free access to water and laboratory chow. Rat pups were delivered spontaneously and reared with their dams. Newborn rat pups were randomly allocated to three groups: normoxia control group (NC, n=10), hyperoxia control group (HC, n=11), and hyperoxia with human UCB-derived MSCs transplantation group (HM, n=12). Rat pups from the NC were initially kept with a nursing mother rat, and after weaning, raised separately in a standard cage kept in room air throughout the experiment. Pups from the hyperoxia groups were maintained with a nursing mother in standard cages in 50 liter Plexiglas chambers, in which the hyperoxic condition (oxygen concentration of 90%) was maintained. Hyperoxia started within 10 hours after birth and continued up to P14. Following this treatment, the rat pups were kept in room air until they were sacrificed at P70. Humidity and environmental temperature was maintained at 50% and 24℃, respectively. Nursing mother rats were rotated daily between litters in the normoxia and hyperoxia groups until P14, to avoid oxygen toxicity. Survival and body weights of rat pups in each group were checked daily until P14, and then checked weekly from P21 to P70. Rats were sacrificed under deep pentobarbital anesthesia (60 mg/kg, intraperitoneal), blood was drawn from cardiac puncture, and organs, including the brain, heart, lung, liver, spleen, were obtained for weighing and histological analyses.

Human UCB-derived MSCs from the 5th passage from a single donor were transplanted at P5. For cell transplantation, 5×10⁵ cells in 0.05 mL phosphate buffered saline (PBS, pH 7.4) were injected intratracheally with a 30-gauge needle syringe. For NC and HC, an equal volume of PBS was administrated intratracheally.5,6 There was no mortality associated with the transplantation procedure.

Organs, including the brain, heart, lung, liver and spleen, were harvested after transcardiac perfusion with ice-cold PBS. Harvested organs were grossly inspected, weighed, and fixed overnight in 10% buffered formalin at room temperature. For morphometric analysis, lungs were fixed in situ by tracheal instillation of 10% buffered formalin at a constant pressure of 20 cm H₂O, and then fixed overnight at room temperature in the same fixative. After tissue processing, fixed organs were embedded in paraffin wax. Four micrometer thick sections were sliced from the paraffin blocks and stained with hematoxylin and eosin. Images of each section were captured with a magnifier digital camera through an Olympus BX40 microscope (Olympus optical Co. Ltd., Tokyo, Japan) and were saved as JPEG files.

The level of alveolarization was assessed by measuring mean linear index (MLI). MLI was defined as the value of the total length of lines drawn across lung section divided by the number of alveoli intercepts encountered, as described by Cooney and Thurlbeck.9 The inflammatory foci of the lung was defined as small pathologic legion which was evidenced by the infiltration of alveolar macrophages, red blood cells, and neutrophils and the thickening of the alveolar wall. Inflammatory foci were scored as normal (no change), minimal (one or two foci recognized under a ×2.5 objective), slight (intermediate between minimal and moderate), or moderate (more than 5, multiple, large inflammatory foci, recognized under a ×2.5 objective) based on modification of previously suggested methods.10-12 The severity of inflammatory foci was operationally defined as follows: normal=0, minimal=1, slight=2, and moderate=3. A minimum of four sections per rat and six fields per section were randomly examined by a pulmonary pathology specialist in a blinded manner.

Immunofluoroscence for angiogenesis (vWF), alveolar macrophages (ED-1) and donor cell localization (human-specific NuMA) was performed on the de-paraffinized 5 µm thick lung sections. To detect antigens, specimens were placed in a solution containing 0.1% (v/v) Triton X-100 and 0.5% (v/v) bovine serum albumin in PBS. The following primary antibody were incubated overnight at 4℃ with 1 : 100 for monocyte/macrophages (CD68, ED-1, mouse monoclonal, hemiproteincon, Millipore, MA, USA), 1 : 50 for NuMA (MERCK, Darmstadt, Germany), and 1 : 200 for vWF (Dako, Glostrup, Denmark) for angiogenesis. Dako polyclonal rabbit anti-mouse immunoglobulins/fluorescein isothiocyanate (FITC) (1 : 200, DakoUk Ltd., Cambridgeshire, UK) and polyclonal swine anti-rabbit immunoglobulins/FITC (1 : 200, DakoUk Ltd., Cambridgeshire, UK) were exposed for 2 hr at room temperature. Vectasheld mount medium with 4', 6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA, USA) was used to preserve staining. Confocal microscopy was carried out at 400X or 800X magnification using Bio-Rad Radiance 2100 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with krypton/argon laser, and images were achieved using the Laser shop 2000 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A minimum of three sections per rat and ten fields per each section were randomly selected and the optical density of immunofluroroscence was measured using the Image J (National Institutes of Health, USA) to assess extent of vWF, and ED-1 positive cells were counted to evaluate alveolar macrophage infiltration.

Withdrawn blood was analyzed for a total WBC count and leukocyte differential and absolute count using the ADVIA 120 Hematology System (Bayer Co., Tarrytown, NY, USA). Lymphocytes were analyzed for anti-CD3 (T cell marker), anti-CD4 (Th cell marker), anti-CD8 (Tc cell marker) using FACSort (Beckton-Dickson Co., San Jose, CA, USA).

Data are expressed as the mean±standard error of the mean. For continuous variables with a normal distribution, a statistical comparison between groups was performed by one-way analysis of variance test with Bonferroni's correction. For variables without a normal distribution, Wiloxon signed-rank tests with Bonferroni's correction were performed. A p-value of <0.05 was considered significant. Stata software (ver. 11.0, Stata Corp LP, College Station, TX, USA) was used for all analyses.

The survival rate during the first 2 weeks of hyperoxic exposure in HC and HM was 82% (9/11) and 83% (10/12), respectively, and no mortality was reported during the recovery period until sacrifice at P70. There was no mortality in the NC throughout the experiment. Birth weight was not different between the experimental groups. Although body weight in HC at P8 and P11 was significantly lower than NC, the final (P70) body weight (377.8±23.0 g, 373.8±21.7 g and 388.8±26.3 g, for the HC, NC and HM, respectively) was not different between groups. In addition, weight of various organs (brain/heart/lung/liver/spleen) including the lung (1.42+0.09 g, 1.45+0.11 g, and 1.55+0.15 g in NC, HC, and HM, respectively) at P70, were not significantly different between the experimental groups.

Fig. 1A presents typical photomicrographs showing histopathological differences of the P70 rat lung in each experimental group. Impaired alveolar growth, as evidenced by fewer and larger alveoli with heterogeneous sizes, was observed in HC as compared to NC, and these impairments in alveolar growth and morphological changes observed in HC were attenuated in HM. Based on morphometric analysis, the MLI, which is indicative of the alveolar size, was significantly higher in HC as compared to NC (p-value <0.05), and this hyperoxia-induced abnormality in morphometry was significantly attenuated in HM (p-value <0.01) (Fig. 1B). There were no grossly or histologically abnormal findings related to hyperoxia or stem cell transplantation, such as hypertrophy, tumor, hemorrhage, hematoma and cancerous pathologic findings in the organs, including the brain, heart, lung, liver and spleen.

Density of immunofluorescence staining for vWF, which indicate the extent of angiogenesis, were significantly reduced in HC as compared to NC in the P70 rat lung (p-value <0.05) (Fig. 2A and B). This hyperoxia-induced decrease in angiogenesis was significantly attenuated in HM (p-value <0.05).

The number of ED-1 positive cells, indicating alveolar macrophages in the lung of P70 rats per high-power field (Fig. 3A), was significantly higher in HC (p-value <0.05) only, not in HM (p-value >0.05), as compared to NC (Fig. 3B). The inflammatory foci in the lungs was graded as normal, minimal, slight and moderate in order to the severity (Fig. 3C). The average severity score of inflammation foci is significantly higher only in HC (p-value <0.05), not in HM, as compared to NC (Fig. 3D).

Donor stem cells were localized with NuMA, a human RNA-specific protein. Although they occur rarely, the expression of NuMA indicating the presence of human UCB-derived MSCs was observed only in HM of the P70 rat lung (Fig. 4).

Compared to NC, the number of total WBC (Fig. 5A) and neutrophil (Fig. 5B) was significantly higher in HC only (p-value <0.05). None of difference was observed in the monocyte counts (Fig. 5C) and lymphocyte counts or its subsets (Fig. 5D) between the experimental groups.