Human MSCs were isolated from waste nasal inferior turbinate and adipose tissues during surgery with the approval of the Institutional Review Board of The Catholic University of Korea (KC08TISS0341, MC16CISI0087, respectively). At passage 4, hTMSCs and AdMSCs were grown in Alpha minimum essential medium (Alpha MEM) (Welgene, Gyeongsan, Korea) or low glucose Dulbecco’s modified Eagle’s medium (Hyclon, GE Healthcare Life Sciences, Little Chalfont, UK), respectively, supplemented with 10% fetal bovine serum, penicillin and streptomycin at 37℃.

All experimental procedures were carried out according to the guidelines of the Institutional Animal Care and Use Committee of The Catholic University of Korea and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Transient focal cerebral ischemia was induced by the intraluminal occlusion of middle cerebral artery (MCAo) as previously described with minor modifications [16]. Briefly, Sprague-Dawley male rats, weighing 270–300 g, were anesthetized with 1.5% isoflurane in a 70% nitrous oxide and 30% oxygen mixture using a face mask. After midline neck incision, a 5-0 silicon-coated nylon monofilament (Doccol Corp., Sharon, MA, USA) was inserted through the external carotid artery and into the internal carotid artery. At 50 minutes after the left MCAo, the filament was withdrawn to allow reperfusion and animals were placed in the incubator. During cerebral ischemia, rectal temperature was maintained at 37℃±0.5℃ using a thermistor-controlled heating blanket. At one day after MCAo, rats were reanesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) cocktails and placed in a stereotaxic frame (David Kopf instruments, Tujunga, CA, USA) for MSC transplantation. After a burr hole was made with a stereotaxic-attached drill, 1.2×10⁵ hTMSCs or AdMSCs/μL in phosphate-buffered saline (PBS) were injected with a Hamilton syringe into the left lateral striatum (0.7 mm anterior to the bregma, 3.2 mm lateral to the midline, and 5.5 mm beneath the dura) in a volume of 5 μL for 5 minutes, and the needle was left for an additional 5 minutes after the injection. Control group was injected with PBS instead of stem cells. Subsequently, dental cement was applied on the skull and the animals were returned to their cages once they become conscious.

Modified neurologic severity score (mNSS), which can evaluate motor (muscle tone and abnormal movement), sensory (visual, tactile, and proprioceptive), and reflex (pinna, corneal, and startle) functions, was used for assessing the efficacy of MSC transplantation. Additionally, limb placement test was performed to check animals’ proprioception and the test results were graded with mNSS (normal, 0; maximal deficit score, 27). To measure MSC-mediated functional recovery accurately, corner test was also performed, which counted the number of each turn direction in front of the corner made by 2 cardboards and calculated the left turn ratio. Tests were performed before and at 1, 7, and 14 days after MCAo surgery.

Infarct volume was assessed at 14 days after MCAo. After cardiac perfusion with normal saline and fixation with 4% paraformaldehyde in 0.1M PBS, the brains were removed and dehydrated with 30% sucrose solution. Then, the brains were cut into 50-μm-thick coronal sections by using a cryostat microtome. Total of 6 sections from each brain were processed for hematoxylin and eosin (H&E) staining. Briefly, sections were stained in hematoxylin solution for 2 minutes, followed by washing with tap water. Then the sections were incubated with eosin for 20 seconds, dehydrated, and mounted. Using an image analysis program (Image J; Bethesda, MD, USA), infarct area was measured by subtracting the area of the noninfarcted hemisphere from that of the contralateral tissue. The percentage of infarct volume was calculated by dividing the sum of the infarct areas by the areas of contralateral hemisphere.

Three coronal sections from each mouse (450-μm interval) were incubated with 10% normal donkey serum for 1 hour and then with the following primary antibodies for overnight at 4℃: rabbit polyclonal anti-doublecortin (anti-DCX, 1:1,000; Millipore Corp., Burlington, MA, USA), mouse monoclonal anti-human nuclei (hNU, 1:200; Millipore Corp.). After washing with PBS, the sections were incubated with the following secondary antibodies for 2 hours at room temperature (22℃±3℃): Alexa488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch). Then, after washing with PBS, the sections were mounted with DAPI (4, 6-diamidino-2-phenylindole)-included mounting media and observed using a confocal microscopy (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).

The data are presented as the mean±standard error of the mean and statistical significance was assessed using IBM SPSS Statistics ver. 21.0 (IBM Co., Armonk, NY, USA) for behavioral tests or GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA) for the rest of statistical analyses. Repeated measures analysis of variance (ANOVA) was performed for the behavioral tests, followed by Duncan post hoc test. ANOVA followed by Duncan post hoc test was performed to compare infarct volume. Student unpaired t-test was performed for immunohistochemistry for hNU and DCX as the data passed Shapiro-Wilk normality test. P<0.05 was considered statistically significant.

As we found functional improvements by hTMSC administration after ischemic stroke, we performed H&E staining for the evaluation of neuroprotective effects. When we measured the infarct size by demarcating eosinophilic dead cell areas (Fig. 2A), hTMSCs- and AdMSCs-treated groups showed the similar infarct volume compared to PBS-administered group (Fig. 2B), suggesting no neuroprotective effects by hTMSC transplantation.

Survival of Grafted hTMSCs Following Ischemic Stroke As a next step, we evaluated the number of surviving MSCs in the brain at 14 days after ischemic stroke. Immunohistochemistry to hNU revealed many hTMSCs and AdMSCs that were labeled with hNU in the infarct area, with a few in the penumbra (Fig. 3A). When we performed a quantitative analysis of hNU-expressing cells, we found no difference in the total number of hNU-positive cells between AdMSCs- and hTMSCs-treated brains, although there was a reducing trend in the hTMSC group (Fig. 3B). Moreover, when we separately counted the number of hNU-expressing cells in the infarct area and the penumbra, there was no difference between the 2 groups (Fig. 3B), suggesting similar survival rates of hTMSCs and AdMSCs after ischemic stroke.

In order to identify molecular mechanisms underlying beneficial effects of hTMSCs after ischemic stroke, we examined hippocampal neurogenesis by staining DCX (Fig. 4A). When we counted the number of DCX-expressing cells in the subgranular zone of the dentate gyrus where new neurons are constantly born, we found that hTMSCs-transplanted group showed a significant increase in the number of DCX-positive cells, compared to PBS-treated group (Fig. 4B). However, there was no difference in the number of hilar ectopic DCX-expressing cells among three groups (Fig. 4C), suggesting that ischemic injury-mediated aberrant hippocampal neurogenesis was not altered by the transplantation of hTMSCs or AdMSCs.