hUCB-MSCs were provided by MEDIPOST Co., Ltd. (Seoul, Korea). This study was approved by the Institutional Review Boards of the Seoul National University Hospital and MEDIPOST Co., Ltd. Umbilical cord blood was collected from the umbilical veins after neonatal delivery with informed consent of the pregnant mother. MSCs were isolated from mononuclear cells (MNCs) using a Ficoll-Hypaque solution (Sigma). Following transfer to α-minimum essential medium (α-MEM; Gibco) supplemented with fetal bovine serum (FBS; Gibco), MSCs were seeded in culture flasks at 5×10⁵ cells/cm². Cells maintained in humidified 5% CO₂ at 37℃ formed colonies of spindle-shaped cells. At 50% confluence, cells were harvested after treatment with 0.25% trypsin-EDTA (Gibco) and were reseeded for expansion.

Adult male Sprague-Dawley rats (250~300 g; Orient, Korea) were housed in an animal care facility under a 12 hours light/dark cycle. Animal care and surgical procedures were performed in accordance with guidelines approved by the Experimental Animals Committee of Seoul National University Hospital for the ethical use of animals. All efforts were made to minimize the suffering and number of animals used in experiments.

Rats were anesthetized using an intramuscular (i.m.) injection of 1% zoletil (20 mg/kg) and xylazine hydrochloride (5 mg/kg). In brief, 1 µl (0.2 µl/min) saline containing 0.2 units of bacterial collagenase (type VII; Sigma) was injected stereotaxically into the striatum at coordinates of 3.0 mm lateral to bregma and 5.0 mm ventral to the cortical surface. After injection, the Hamilton syringe was left in place for 5 minutes. The needle was slowly removed after an additional 10 minutes to prevent backflow. The hole was sealed with bone wax and the wound was sutured.

Two days after injection of collagenase, we performed behavioral tests and divided the rats into 2 groups. Rats in the control group were injected with 5 µl of 10 X PBS. For transplantation in the experimental group, 5×10⁵ MSCs in 5 µl of PBS were stereotactically injected into the left lateral ventricle of anesthetized animals at 3.0 mm lateral to bregma and 5.0 mm ventral to the cortical surface. Animals were not given any prophylactic immunosuppressant.

Behavioral tests were performed and evaluated by the rotarod and limb placement tests 1 day before ICH and at 2, 9, 16, 30 days after ICH (Fig. 1A).

In the rotarod test, rats were trained at a constant speed of 16 rpm (600 seconds) for 3 days before ICH. The rats were placed on an accelerating rod (speed from 3.5 to 35 rpm) and the duration (s) the rats remained on the rotarod was measured three times, 1 day before surgery. Rotarod test data were presented as percentiles of the maximal duration compared with the baseline control (before ICH).

The limb placement test had three tasks that assessed motor integration of the forelimbs and hindlimbs by checking responses to tactile and proprioceptive stimulations. 'Visual forward' was the observation of forelimb flexion when the tail was held up. The stretch of the forelimbs towards the table was evaluated as normal stretch (0 point) and abnormal flexion (1 point). 'Visual lateral' was the observation of the forelimb stretch by stimulating the rat's whiskers while the examiner held the rat's trunk. The evaluations were: normal lifting, 0 point; abnormal lifting, 1, 2, or 3 points according to the number of normal stretches. 'Proprioception' was the observation of stepping up of the forelimbs and hindlimbs on the table after a pull-down of the forelimb and hindlimbs below the level of the table (normal lifting, 0 point; abnormal lifting, 1, 2, or 3 points according to the number of normal stretches). The authors defined the severity of injury by scoring on a scale of 0 to 10 (normal score 0, maximal deficit score 10).

When behavioral tests were completed at 30 days after ICH, the animals were anesthetized and perfused intracardially with saline followed by ice-cold 4% paraformaldehyde in PBS (pH 7.4) for 10 min. Brains were removed, post-fixed for one day at 4℃, and then cryoprotected overnight in the same fixative supplemented with 25% sucrose. The tissues were embedded in OCT compound (Sakura Finetek, Inc., Tokyo, Japan) and frozen at -70℃ with dry ice. Coronal sections (14 µm thick; 28 µm apart) were cut with a cryostat (Leica Microsystems, Nussloch, Germany). Sections were stained with Hematoxylin and Eosin (H&E) and the volume of lesion (e.g., cavity, cellular debris) was calculated using ImageJ 1.38× (National Institutes of Health) as:

Volume of lesion (mm³)=area of lesion (mm²)×thickness of slice (mm/slice)×sampling interval (slice)

The sections were permeabilized with 0.1% saponin for 30 min and blocked with 5% normal goat serum (NGS) for 30 min. The sections were also permeabilized for 30 min with 0.1% Triton X. Thy were then incubated overnight at 4℃ with primary antibody followed by incubation in fluorescently labeled secondary antibody and mounted with Vectashield medium containing DAPI (Vector Laboratories, Burlingame, CA). The primary antibodies used in the experiments were: anti-Human Nuclei (1:100; Chemicon), anti-laminin (1:200; Sigma, Inc.), anti-neuron specific beta III tubulin (Tuj1) (1:100; abcam), anti-COX2 (1:100; abcam), anti-TNF alpha (1:100; abcam), anti-CD11b (1:100; BD Pharmingen), and anti-myeloperoxidase (1:100; abcam). All secondary antibodies, Alexa 488, Alexa 594 goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Molecular Probes (Invitrogen, Co.). The In Situ Cell Death Detection Kit, TMR red (Roche), was used for indirect the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) staining.

Fluorescently immunolabeled sections were imaged by confocal laser scanning microscope (Leica TCS SP8; Leica Microsystems, Mannheim, Germany). Tile scanning was performed (10×, 20× magnification) following which the images ware merged to obtain the whole brain image. To evaluate the labeling, confocal scanning was performed at 40× magnification. Staining density was analyzed using Image-Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD, USA).

RNA was isolated at day 5 after ICH using the RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany). First-strand cDNA synthesis was carried out by random priming of the total RNA using a random primer mixture (Invitrogen) and reverse transcriptase with superscript 3 (Invitrogen). The sequences of the primers and the cycles of PCR are given in Table 1. The PCR products were resolved in 1% agarose gels. The band density from gel images was determined by Gel Doc XR System (Bio-Rad, Milan, Italy). Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Animals were sacrificed to extract the brain at 5 days after ICH and whole brains were divided into ipsilateral (infarcted) and contralateral hemispheres, which were frozen in liquid nitrogen. Brain tissues from each group were homogenized in Neuronal Protein Extraction Reagent (Pierce), which included 1× protease and phosphatase inhibitor (Pierce). Protein concentrations were determined using BCA Protein Assay Kit (Pierce). The samples were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham) that were blocked in 5% non-fat dried milk (NFDM) and incubated with anti-COX2 (1:1000; abcam), anti-TNF alpha (1:1000; abcam), anti-CD11b (1:500; BD Pharmingen), and anti-myeloperoxidase (1: 1000; abcam) and β-actin. Membranes were incubated for 1 hour with a secondary antibody and the intensity of all protein bands was quantified by densitometry using the Bio-image Processing System (GenDix). Densitometric values were normalized to those of beta-actin, used as an internal control.

Data are presented as mean±SEM. The significance of differences between test conditions was assessed using the Mann-Whitney U test. Comparisons of continuous variables between the two groups were performed by a two-way ANOVA. p<0.05 was considered statistically significant. The GraphPad Prism 5 software (GraphPad Prism, Sandiego, California, USA) was used for the statistical tests.

Hemorrhagic lesions mainly appeared in the striatum as observed by H&E staining at 4 weeks after transplantation. Ventricles were expanded in the ipsilateral hemisphere more than in the contralateral (Fig. 1B). The volume of lesion in the hUCB-MSCs-treated group was significantly smaller than the controls (Fig. 1C, ^**p<0.01). These results indicate that hUCB-MSCs reduce the volume of lesion after ICH.

To evaluate behavioral outcome, the rotarod and limb placement tests were performed (Fig. 1A). The hUCB-MSCs-treated group could remain on the rotarod for a significantly longer duration than the controls at 4 weeks after transplantation (Fig. 1D, ^****p<0.001). Additionally, at 4 weeks after transplantation, the limb test score revealed a significant neurological benefit in the hUCB-MSCs-treated group compared with the controls (Fig. 1E, ^**p<0.01). These results indicate that hUCB-MSCs promote functional recovery after ICH.

Previously, we observed neurogenesis, angiogenesis, and anti-apoptosis effects of hUCB-MSCs in rat ischemia models. To determine if transplantation of hUCB-MSCs might have a similar impact on in the post-hemorrhagic brain, we performed immunofluorescence staining for specific markers: Tuj1 for neurons and Laminin for blood vessels, and TUNEL staining for apoptotic cells in the rats at 4 weeks after transplantation. We found Tuj1-positive cells (green) in the subventricular zone (SVZ) or in the hemorrhage area (Fig. 2A). Statistical analysis indicated an increase, although not significant, in the number of Tuj1-positive cells in the hUCB-MSCs-treated group than in the controls (Fig. 2D). Laminin-positive cells (green) were observed in the hemorrhage site and were present in large numbers in the peri-infarct regions (Fig. 2B). The number of laminin-positive cells was significantly higher in the hUCB-MSCs-treated group than in the controls (Fig. 2E, ^*p<0.05). These results indicate that hUCB-MSCs promote angiogenesis after ICH. TUNEL-positive cells (red) were found within the hemorrhage lesion as well as in the surrounding periphery in the hUCB-MSCs-treated rats and the controls (Fig. 2C). Although the number of TUNEL-positive cells was lower in the hUCB-MSCs-treated group than in the controls, the difference was not significant (Fig. 2F).

To confirm the existence of transplanted hUCB-MSCs in the rat brains, we performed immunofluorescence staining with anti-human nuclei antibody (HuNu). HuNu-positive hUCB-MSCs (red) survived and majority of the cells were distributed close to the hemorrhagic boundary zone at 3 days after transplantation (Fig. 3A). However, HuNu-positive cells were not detected in both groups at 4 weeks after transplantation (data not shown).

To investigate the effect of hUCB-MSCs in the inflammatory response, the expressions of inflammatory factors (tumor necrosis factor alpha [TNF-α], cyclooxygenase [COX]-2, CD11b positive to microglia, and myeloperoxidase [MPO] positive to neutrophils were examined at 3 days after transplantation.

Confocal micrographs of brain sections showed the expression of TNF-α, COX-2, CD11b, and MPO in peri-hemorrhagic regions (Fig. 3B~E). The expression levels of these inflammatory factors were lower in the hUCB-MSCs-treated rats than in the controls. However, there was no significant difference between the two groups (Fig. 3F~I).

The mRNA levels of TNF-α, COX-2, microglia, and neutrophils were detected by RT-PCR (Fig. 4A). The expression levels of these inflammatory factors were down-regulated in the hUCB-MSCs-treated rats compared with the controls. However, there was no significant difference between the two groups (Fig. 4B~E).

The protein levels of the inflammatory factors were detected by western blot (Fig. 4F). The protein expression levels were down-regulated in the hUCB-MSCs-treated rats compared with the controls. However, there was no significant difference between the two groups (Fig. 4G~J).