Adult male Sprague-Dawley rats (260~300 g; Orient bio, Korea) were housed in an animal care facility under a 12 h light/dark cycle, with food and water available ad libitum. Animal care and surgical procedures were carried out in accordance with guidelines on the ethical use of animals approved by the Experimental Animals Committee of Seoul National University Hospital. All efforts were made to minimize the number of animals used and their suffering. Animals were divided into 4 groups (n=48): one control group (n=12) and 3 experimental groups (n=12 per group, total n=36), which were sacrificed post-MCAO.

Rats were anesthetized by an intramuscular injection of Rompun (10 mg/kg of xylazine hydrochloride, Bayer, Leverkusen, Germany) and Zoletil 50 (75 mg/kg of zolazepam and tiletamine, Virbac, Carros, France). Rectal temperature was maintained at 37℃ using a thermistor-controlled heating blanket. Transient MCAO was induced using an intraluminal vascular occlusion method, as previously described [12]. Briefly, a 2 cm incision was made at the center of the neck; the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed under an operating microscope. A 4/0 monofilament nylon thread whose tip was rounded by heating was advanced into the lumen of the ICA until it blocked the origin of the MCA. One and half hours after MCAO, animals were re-anesthetized for reperfusion, which was performed by suture withdrawal. After recovery from anesthesia, animals were allowed free access to food and water. After surgery, 28 of the 48 animals survived; consequently, a total of 28 rats were randomly assigned to the control and experimental groups.

This study was approved by the Institutional Review Board of MEDIPOST Co., Ltd. UCB-MSCs were separated and maintained as described previously [13]. Umbilical cord blood was collected from the umbilical veins after neonatal delivery, with informed consent from the pregnant mothers. MSCs were isolated by separating mononuclear cells (MNCs) using a Ficoll-Hypaque solution (d=1.077 g/cm³; Sigma, St. Louis, MO, USA). MNCs were transferred into a-minimum essential medium (a-MEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, USA). They were then seeded in culture flasks at a density of 5×10⁵ cells/cm². Cells were maintained in a humidified 5% CO₂ chamber at 37℃ and colonies composed of spindle-shaped cells were formed. Once cells were 50% confluent, they were harvested after treatment with 0.25% (w/v) trypsin-EDTA (Gibco) and were reseeded for expansion. Three different hUCB-MSCs were prepared and they differed based on only the original UCB donor.

The prepared cells were assigned to either a control group or 1 of the 3 experimental groups. Group 1 rats (control) were administered 1X PBS intraparenchymally 48 h after MCAO. Group 2 rats were injected intraparenchymally with 5 µl total volume of hUCB-MSCs (G19, 5.0×10⁵ in 1XPBS) 48 h after MCAO. Group 3 rats were injected intraparenchymally with 5 µl total volume of hUCB-MSCs (G16, 5.0×10⁵ in 1XPBS) 48 h after MCAO. Group 4 rats were injected intraparenchymally with 5 µl total volume of hUCB-MSCs (G5, 5.0×10⁵ in 1XPBS) 48 h after MCAO.

Limb placement tests (LPTs) included 8 subtests and were performed 2 days after ischemia, as previously described [14]. Briefly, the test consisted of 3 domains: (1) visual forward, (2) visual lateral, and (3) proprioception. Visual forward was defined as the observation of forelimb flexion when holding the animal by the tail. The stretch of the forelimbs towards the table was evaluated as either a normal stretch (0 points) or abnormal flexion (1 point). Visual lateral was defined as the observation of a forelimb stretch after a stimulus was applied to the rat's whiskers when it was held by its trunk. The visual lateral was scored as follows: normal lifting (0 points) or abnormal lifting (1, 2, or 3 points, according to the times of normal stretch). Proprioception was defined as the observation of stepping up of the forelimb and hindlimb on the table after pulling down the forelimb and hindlimb below the level of the table. This occurred 3 times and was scored as normal lifting (0 points) or abnormal lifting (1, 2, or 3 points, according to the times of normal stretch). Therefore, when all 3 domains were assessed, the highest possible score was 10.

The rotarod test (Panlab, Barcelona, spain) consisted of an accelerating rod, which provided an index of forelimb and hindlimb motor coordination and balance [15]. The rat was placed on an accelerating rod, whose velocity was slowly increased from 4 rpm to 40 rpm within a 10 min period. The duration for which the rat remained on the accelerating, rotating rod was measured in seconds. A trial was terminated when the rat fell off the rotarod. The animals were trained with 3 trials per day for 3 days before MCAO to obtain stable baseline values. The rotarod data are presented as a percentage [(mean duration of the 3 post-MCAO trials/ duration of pre-MCAO baseline trials) ×100].

After anesthesia, the animals were intracardially perfused with saline, followed by ice-cold 4% paraformaldehyde (PFA; in PBS) for 10 min. Their brains were removed, post-fixed for 1 day at 4℃, and then transferred sequentially at one-day intervals into 10%, 20% and 30% sucrose until they sank to the bottom of the container. The tissues were embedded in OCT compound (Sakura Finetek, Inc., Torrance, CA), and frozen at -70℃ using dry ice. The brain sections were cut to a thickness of 30 µm with a cryostat (Leica CM 3000, Leica, Solms, Germany).

The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to measure apoptotic cell death in the control and experimental groups. Using an in situ cell death detection kit (TMR Red; Roche Diagnostics, Mannheim, Germany), the frozen tissue section slides were washed 3 times with 1X PBS and permeabilized for 2 min on ice. The permeabilized tissues were washed 3 times with 1X PBS; 50 µl of TUNEL reaction mixture was added to each tissue. The treated tissues were then incubated at 37℃ in a humidified environment for 1 h. After incubation, the tissues were washed 3 times with 1X PBS and imaged by fluorescent microscopy.

Fluorescent immunolabeled sections were imaged with a Meta confocal microscope (LSM model 510; Carl Zeiss MicroImaging, Inc., Jena, Germany) equipped with 4 lasers (Diode 405, Argon 488, HeNe 543 and HeNe 633). Each channel was separately scanned using a multitrack-photomultiplier tube (PMT) configuration to avoid bleeding between the fluorescent labels in order to visualize the labeled structures in relation to other cells.

The infarct size was measured with Image J (NIH, v1.38). The sections were scanned with a high-resolution scanner (Epson Perfection V700 photo), and the 2 hemispheres were compared using ImageJ. The infarct lesion was drawn using a graphic tablet tool and the size was summed and presented as a percentage of the size of the intact hemisphere. Infarct Size = lesion size / non-lesion size ×100.

Statistical analysis was performed using Graph-Pad Prism version 3.03 (GraphPad Software. Inc., San Diego, CA). All data are presented as mean±SEM. A repeated-measures analysis of variance (ANOVA) was performed on all behavioral data. The quantification of the immunofluorescent staining in the control and experimental groups was performed using the Student's t-test.

Abnormal motor performance caused by motor cortex infarcts was assessed by the modified LPT and rotarod. Each experimental group was administered a different lot number of hUCB-MSCs (e.g., G5, G16, G19) and it was compared to the control group. There were no significant differences in the neurological outcomes before cell transplantation. However, there was significant functional improvement in both motor tests in the G19 and G5 hUCB-MSC-treated groups starting 7 days after cell injection (therefore, 9 days after MCAO surgery) compared to the control group (rotarod and LPT: ^*p<0.05; Fig. 1A). There was no improvement in the G16 hUCB-MSC-treated group compared to the G19 and G5 hUCB-MSC-treated groups in either the rotarod (Fig. 1A) or the LPT (Fig. 1B). Therefore, motor dysfunction can be improved with hUCB-MSC administration without regard to the donor type.

To determine whether hUCB-MSCs could reduce the infarct size in the ischemic brain, brain tissue was stained with cresyl violet (Nissl staining). Infarcted lesions were mainly concentrated in the cortex and striatum at 28 days after hUCB-MSC injection. The entire ischemic tissue area was transformed into cysts. The relative infarct size in all hUCB-MSC- treated experimental groups was significantly smaller than that in the control group (^*p<0.05; Fig. 1C and D). Therefore, hUCB-MSC administration can reduce stroke-induced infarct size.

To determine whether donor-dependent hUCB-MSC administration enhanced angiogenesis in endothelial cells or endogenous astroglia, we performed double-labeled immunohistochemistry with GFAP and anti-human nuclei, and laminin and anti-human nuclei. hUCB-MSC treatment increased the percentage of GFAP- and laminin-positive cells in the ischemic boundary zone at 28 days after MCAO ischemic injury compared to the control group (Fig. 2A, B and 3A, B). There was no additional difference in the percentage of GFAP- and laminin-positive cells between the G19, G16, and G5 hUCB-MSC-treated groups. Co-localization of anti-human nuclei and GFAP or laminin was confirmed through double labeled immunofluorescence. GFAP expression was limited to a cell population in the border zone of the ischemic cortex. Expression of anti-human nuclei was located near GFAP and laminin positive populations of cells.

It was possible to determine approximately the mount through a measure of Human Nuclei (HuNu) positive cells. On average, on the basis of the measured mean value in the 1 mm×1 mm square, the number of surviving cells in each group was 88.8×10³±1023 (G19), 33.6×10³±2012 (G16), and 55.2×10³±1286 (G5). The equivalent value based on the amount of administered cells was 17.76% (G19), 6.72% (G16), and 11.04% (G5).

To determine whether hUCB-MSC administration induced neurogenesis in a donor-dependent manner, we performed immunofluorescent staining of nestin and GFAP-delta (Fig. 4A). Nestin and GFAP-delta expression was primarily located throughout the brain in the ischemic boundary zone. The sections were analyzed with Image Pro Plus software. The number of cells positive for nestin and GFAP-delta, markers of neurogenesis, was significantly increased from the ipsilateral striatum to the ischemic striatum in all hUCB-MSC-treated groups compared to control group (Fig. 4B). There were no differences between the G19, G16, and G5 hUCB-MSC-treated groups. Furthermore, hUCB-MSCs significantly reduced the density of apoptotic cells in the ischemic brain regardless of the donor, as indicated by the reduced number of TUNEL-positive, DNA-fragmented cells (Fig. 4C).