Flow Cytometry

The levels of CD105 and CD73 (MSC surface marker) expression in P2 and P6 MSC were evaluated using flow cytometry (FACScan; Becton-Dickinson, Rurtherford, NJ), as previously described (Bang et al., 2005). The CD105 and CD73 antigen were detected using un-conjugated monoclonal anti-human Endoglin (CD105, R&D systems, Minneapolis, MN) and phycoerythrin-conjugated anti-CD73 monoclonal antibody (BD Biosciences).

To compare observe the mobilization ability between P2 and P6 MSCs, cells were stained with mouse monoclonal anti-human CXCR4 (Abcam, Cambridge, MA), followed by anti-mouse IgG fluorescein isothiocyanate (FITC; 1:400; Molecular Probes), according to the manufacturer`s instruction.

adhesive-removal somatosensory test (Chen et al., 2001b; Chen et al., 2004; Li et al., 2002), two small pieces of adhesive-backed paper dots (of equal size, 113.1 mm²) are used as bilateral tactile stimuli occupying the distal-radial region on the wrist of each forelimb. The time to remove each stimulus from forelimbs is recorded on five trials per day. Before surgery, the animals are trained for 3 days. If the rats are able to remove the dots within 10 s, they were subjected to surgery. The time to remove the dot is recorded. Using modified Neurological Severity Score (mNSS), neurological function was graded on a scale of 0-18 (normal score, 0; maximal deficit score, 18). The mNSS is a composite of the motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), reflex , and balance tests (Chen et al., 2001b; Li et al., 2002).

7. MRI Studies and Measurement of Infarct Volume

Rats were anesthetized with i.p. ketamine (50 mg/kg) and xylazine (6 mg/kg). Each rat was placed in an animal holder/MRI probe apparatus and positioned inside the magnet. The animal’s head was held in place inside the QUAD Wrist (GE Healthcare, Milwaukee, WI) imaging coil. All MRI measurements were performed using a 1.5-T, 60-cm-bore superconducting magnet (Signa EXCITE MR; GE Healthcare). I used T2-weighted images (T2-WI), as previously reported (Nomura et al., 2005), which were obtained from a 2.0-mm-thick coronal section, with no gap using a 40×40 mm field of view, a repetition time of 4100 ms, an echo time of 40 ms, and a b-value of 0. Images were reconstructed using a 256 × 256 image matrix. MRI measurements were obtained 1 day and 14 days after tMCAo (n = 4 for each group). For hMSC-treated groups, cells were intravenously delivered immediately after

the initial MRI.

The ischemic lesion area was calculated from T2-WI using the PACS (Picture Archiving and Communications System) imaging software (PiViewSTAR version 5.0; INFINITT, Seoul, South Korea). For each slice, the higher intensity lesions in T2-WI, where the signal intensity was 1.25 times higher than the counterpart in the contralateral brain lesion, were marked as the ischemic lesion area and infarct volume was calculated taking slice thickness (2 mm/slice) into account.

8. Histological and Immunohistochemical Assessment

8.1. TTC Staining and Quantitative Analysis of Infarct Volume

The brains were removed immediately and sectioned coronally into six slices (2 mm thick) using a rodent brain matrix (Harvard Instrument Inc., South Natick, MA). Brain slices were placed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Chemical, St. Louis, MO) in the dark and incubated at 37°C for 30 min. The brain slices were then removed from the incubator. The cross-sectional area of infarction in each brain slice was examined under a dissection microscope and measured using image analysis software (TINA 2.0; Raytest, Straubenhardt, Germany). The total infarct volume for each brain was calculated by summing the infarcted area of all brain slices.

paraformaldehyde solution. The brains were removed quickly and kept in 4%

paraformaldehyde solution overnight at 4°C, embedded in 30% sucrose solution until they sank, and frozen sectioned on a slidingmicrotome in 30-µm-thick coronal sections. To count BrdU-positive cells in the left subventricular zone (SVZ) and the ischemic boundary zone (IBZ), systematic random sampling was conducted, as previously described (Prockop et al., 1997). Every sixth section in a series of 60-µm-thick coronal sections between bregma levels +1.0 and –1.0 mm (total of six sections per brain) were collected to quantify BrdU labeling.

I counted BrdU-positive cells to evaluate the degree of neurogenesis. To do so, DNA was first denatured by incubating brain sections in 2 N HCl at 37°C for 1 h. The sections were then rinsed with Tris buffer and treated with 0.3% H₂O₂ to block endogenous peroxidase. The sections were blocked with 10% normal horse serum and 0.1% Triton X-100 in PBS for 30 min at room temperature and incubated with a mouse monoclonal antibody (mAb) against BrdU (1:200; Roche) overnight at 4°C. The sections were then incubated with horse anti-mouse IgG for 1 h at room temperature and with an avidin–biotin–peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame,CA) for 1 h at room temperature and developed in diaminobenzidine (DAB) substrate-chromogen (DAB kit;

DakoCytomation, Glostrup, Denmark). The tissues were rinsed in 0.1 M PBS to stop the DAB reaction. The labeled tissue sections were then mounted on gelatin-coated slides and analyzed under a bright-field microscope (E600; Nikon, Tokyo, Japan).

8. 3. Stereological Cell Counts

To determine the number of BrdU-labeled cells in the SVZ and IBZ of the ipsilateral

hemisphere, every 6^th section (each 60 µm) between bregma levels +1.0 and –1.0 mm was selected (total of five sections per brain). The unbiased stereological estimate of the total number of BrdU-immunopositive cells inthe SVZ and IBZ was made using the optical fractionator technique (Plane et al., 2004). This sampling technique is not affected by tissue volume changes and does not require reference volume determinations(West et al., 1991).

Sampling was performed with the Computer-Assisted Stereological Toolbox (CAST) system (version 2.3.1.5, Olympus Denmark A/S, Ballerup, Denmark), using an Olympus BX51 microscope. The SVZ and IBZ were delineated using a 1.25× objective, which generated counting areas of 84.62 × 84.62 µm. A counting frame (3,580 µm²) was placed randomly on the first counting area and moved through all the counting areas systemically until the entire delineated area was sampled. The sampling frequency was chosen so that 100–200 BrdU-positive cells were counted in each specimen in the ipsilateral hemisphere. Actual counting was performed using a 100× oil objective lens. Guard volumes (4 µm from the top and 4–6 µm from the bottom of the section) were excluded from both surfaces to avoid the problem of lost caps, and only the profiles that came into focus within the counting volume (with a depth of 20 µm) were counted. The total number of BrdU-positive cells was estimated using the optical fractionator formula (West et al., 1991).

For double-immunofluorescence staining, the tissues were washed three times with PBS and nonspecific binding was blocked with 10% normal horse serum. Then, the tissues were treated overnight at 4°C with monoclonal antibody specific for human nuclei matrix antigen (NuMA; Oncogen, Seattle, WA) diluted 1:100 in PBS. Following sequential incubation with anti-mouse Alexa 594 (1:400; Molecular Probes, Eugene, OR), secondary antibody was bound to the first antibody to NuMA. Cells derived from hMSCs were identified using morphologic criteria and immunohistochemical staining with NuMA (present in the donor cells, but not present in the parenchymal cells).

To visualize the cellular colocalization of NuMA and cell-type-specific markers in the same cells, each coronal slide was treated with the first primary antibody to NuMA, as described above, and then incubated with glial fibrillary acidic protein (GFAP for astrocytes;

Sigma, St. Louis, MO), the neurofilament subunit (NF-L for neurons; Chemicon, Temecula, CA), and doublecortin (DCX C-18 for migrating neurons; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and then incubated with anti-rabbit Alexa 488 (1:400; Molecular Probes), anti-mouse Alexa 488 (1:400; Molecular Probes), or anti-goat FITC (1:200; Vector Laboratories) antibody for 1 h at room temperature. The sections were washed in PBS, rinsed with double-distilled water, and mounted on slides using an anti-fade mounting medium (Permafluor; Molecular Probes).

To determine whether BrdU-labeled cells differentiate into neuronal or astroglial cells, sections were treated with 2 N HCl and rinsed as above, blocked with 10% normal horse or goat serum and 0.1% Triton X-100 in PBS for 30 min at room temperature, and incubated with rat monoclonal anti-BrdU (1:100; BD Biosciences, San Jose, CA) overnight at 4°C.

After rinsing with primary antibodies, the sections were incubated for 1 h with cy3-conjugated goat anti-rat IgG (1:400; Abcom, Cambridge, UK). The sections were then incubated with NF-L, neuronal nuclear protein (NeuN for neuron; Chemicon), DCX, and GFAP overnight at 4°C, and incubated with anti-rabbit Alexa 488 (1:400), anti-mouse Alexa 488 (1:400), or anti-goat FITC (1:200) antibody for 1 h at room temperature. The sections were washed in PBS, rinsed with double-distilled water, and mounted on slides using an anti-fade mounting medium (Permafluor, Molecular Probes). The specimens were examined on a Zeiss LSM510 confocal imaging system (Carl Zeiss).

To assess what percentage of newborn cells differentiated into a neuronal phenotype after hMSC treatment, the double-positive percentage was calculated from the number of double-positive cells and the total number of BrdU-positive cells. The mean values were calculated from eight slides from four rats, each slide randomly containing eight fields from the hemisphere.

9. ELISA for Growth Factor

Tissue extracts from ischemic hemispheres (n = 4 for each group) were prepared by homogenization and lysis with 25 mmol/L Tris, 1% Triton X-100, 0.5 mmol/L EDTA, 150 mmol/L NaCl, and a protease inhibitor cocktail (Calbiochem, Darmstadt, Germany). Total

protocol (R&D Systems). I also performed an ELISA test for recombinant murine SDF-1 to evaluate the level of expression of the chemokine SDF-1 in the ischemic hemisphere (R&D Systems). Each sample was analyzed in triplicate.

10. Statistical Analysis

Statistical differences between groups were evaluated using one way analysis of variance. P values <0.05 were deemed statistically significant.

III. RESULTS

1. Characteristics of hMSC

To determine possible differences in viability, depending on the passage of the hMSCs, cell viability of P2, P6, and P15 hMSCs was measured by observing the amount of MTT reduction; no significant difference was detected between P2 and P6 hMSCs (Fig. 1A).

Similar results were obtained when cell viability was assessed with the trypan blue dye exclusion assay; both P2 and P6 showed over 95% viability.

Flow cytometric analysis (Fig. 1B) indicated that both P2 and P6 cells had high levels of expression of stem cell markers, CD105 (median 90.3%, range 83.1–96.4% for P2; median 91.7%, range 77.1–99.7% for P6) and CD73 (median, 98.0%, range 97.4–98.8% for P2;

median 94.3%, range 83.8–99.9% for P6). No significant difference was found between P2 and P6 MSCs. These cells did not express CD34 or CD45 antigen (data not shown).

Fig. 1. Characteristics of MSCs. (A) Viability of P2, P6, and P15 hMSCs. Cells were cultured and MTT reduction was serially monitored by optical density at 540 nm. The ordinate represents % of cell survival as measured by MTT assay. The day 1 cell viability values obtained was normalized to 100%. Numbers presented are averages ± standard errors.

Some error bars were too small to appear on the graph. (B) Flow cytometric analysis of MSC surface markers, CD105 (upper lane) and CD73 (lower lane), of P2 and P6 hMSCs.

2. Neurologic functional testing

At day 14 after tMCAo, rats that received P2 or P6 hMSCs showed functional recovery compared to the tMCAo-only group (Fig. 2A, B). However, the degree of improvement differed, depending on the passage of the hMSCs received. On the adhesive-removal test, rats that received P2 hMSCs showed a higher level of functional improvement than rats receiving P6 hMSCs (P<0.05). In the same manner, neurological deficits at 14 days after stroke were less severe in the rats that received P2 hMSCs than in those receiving P6 hMSCs (P<0.05) or rats in the tMCAo-only group on the mNSS test (P<0.001).

3. Ischemic lesion size measurement by MRI and TTC analysis

An estimate of lesion size was obtained using both MRI (24 h and 14 day) and TTC staining (14 days after tMCAo) (Fig. 2C). The T2-WI lesion volume at 24 h after tMCAo was similar among the groups. Although a trend was observed in the hMSC-treated group for lesion volumes to be smaller at 14 days after tMCAo compared to those in the tMCA-only group, the difference was not statistically significant (P>0.05). The T2-WI lesion volume was 217 ± 18 mm³ in the tMCAo-only group, 179 ± 20 mm³ in the P6 hMSC-treated group, and 184 ± 19 mm³ in the P2 hMSC-treated group, while the TTC infarct volume in the three groups was 143 ± 16, 122 ± 14, and 129 ± 13 mm³, respectively.

Fig. 2. Results of behavioral tests and infarct volume measurements. Adhesive-removal test (A) and modified neurological severity score (mNSS) test (B), before and after transient middle cerebral artery occlusion (tMCAo). Open circle – tMCAo-only group, filled circle – P6 hMSC-treated group, and open triangle – P2 hMSC-treated group. (C) and (D) Evaluation of ischemic lesion volume with T2-weighted image and TTC staining in rats of (a) the tMCAo-only group, (b) P6 hMSC-treated group, and (c) P2 hMSC-treated group. T2 -weighted image obtained at 24 h (column 1) and 14 days (column 2) after tMCAo. The 24 h images were obtained just before intravenous cell or vehicle injection. Brain slices were stained with TTC to visualize lesions 14 days after tMCAo (column 3). The coronal forebrain sections were obtained at the level of the caudato–putamen complex. (Scale bar =

500 μm). (D) Meserment infarct size of T2-weighted image and TTC staining image at 1day and 14days after tMCAo.

**P<0.01, the P6 hMSC-treated group versus the tMCAo-only group.

***P<0.001, the P2 hMSC-treated group versus the tMCAo-only group.

# P<0.05, the P2 hMSC-treated group versus the P6 hMSC-treated group.

4. Cell proliferation in the SVZ of the ischemia brain

In the sham group, a few BrdU-positive cells were concentrated in the SVZ (Fig. 3A), while in the tMCAo groups, BrdU-labeled cells were more widely dispersed throughout the SVZ (Fig. 3B). In the P2 and P6 hMSC-treated groups, increased numbers of more widely distributed BrdU-positive cells were observed, involving the enlarged SVZ, corpus callosum, and striatum, mainly at the IBZ (Fig. 3C, D).

Unbiased stereological analysis was used to quantify cell proliferation in the SVZ and IBZ at 14 days (Fig. 3E, F). BrdU-labeled cells in the SVZ were counted in a clearly defined region by outlining the dorsolateral SVZ and IBZ. Both ischemia and hMSC stimulated cell proliferation in the SVZ and IBZ on the ischemic side (P<0.001 in both cases). The most BrdU-positive cells were in the hMSC-treated group and the fewest in the sham operation group; the number in the tMCAo-only group was in between. The numbers of BrdU-labeled cells increased significantly in the hMSC-treated groups over the sham operation group or the PBS-treated tMCAo group (P<0.01 in all cases). Of the hMSC-treated groups, BrdU-labeled cells in the SVZ increased significantly in the P2 hMSC-treated group compared to the P6 hMSC-treated group (P<0.01). A similar trend was observed in the IBZ (Fig. 3F).

Fig. 3. Comparison of neurogenesis between the groups. BrdU immunostaining in the subventricular zone of the ipsilateral hemisphere at day 14 in each group: (A) sham operation, (B) tMCAo + PBS, (C) tMCAo + P6 MSC, and (D) tMCAo + P2 MSC. In the inner box, only secondary antibody without BrdU was used as background control (negative

5. Identification and characterization of donor and newly developed cells.

Labeling with NuMA-positive cells (~3–5% of 1 × 10⁶ hMSCs) survived and were observed in multiple areas of the ipsilateral hemisphere, including the cortex and striatum. Most NuMA-labeled hMSC (~50–70% of the total) were located in the ischemic boundary zone.

hMSC themselves were unlikely to proliferate after the time of infusion because no NuMA-labeled hMSC was BrdU-positive (Fig. 4). Few NuMA-positive cells were double-NuMA-labeled with the migrating neuronal marker DCX (~5% of the total; Fig. 4C), and no NuMA-positive cells double-labeled with GFAP (Fig. 4A) or NF-L were detected (Fig. 4B).

Fig. 4. Phenotype of NuMA-labeled cells in the boundary zone 14 days after tMCAo.

BrdU- and NuMA-positive cells double-labeled in the subventricular and boundary zone. No NuMA-positive cells merged with BrdU-positive cells. No NuMA-positive cells expressed GFAP or NF-L (C). Some NuMA-positive cells expressed DCX (D). Scale bar: 100 µm (A, B); 20 µm (C, D).

In contrast, some BrdU-positive cells in the ipsilateral striatum and cortex were reactive for the neural or glial markers used. A significant proportion of BrdU-labeled cells expressed DCX, GFAP, NeuN, and NF-L (Fig. 5). As shown in the Table 1, the proportions of DCX-, GFAP-, NeuN-, and NF-L-expressing BrdU-positive cells were higher in the hMSC-treated groups than in the other groups. Most of the BrdU-positive cells in groups P2 and P6 exhibited neuronal or glial phenotypes. However, no significant difference was observed in the proportion of BrdU-positive cells expressing neuronal or glial phenotypes between groups P2 and P6 (P>0.05 in all cases).

Table 1. Quantitative analysis of BrdU-labeled cells and their phenotypes.

Groups, Mean ± SD

Per section Sham MCAo+PBS MCAo+P6 hMSC MCAo+P2 hMSC

Total BrdU⁺ cells 56 ± 12 249 ± 14 855 ± 14 947 ± 18 BrdU⁺ DCX⁺ cells 0.2 ± 0.1 (0.5) 32.8 ± 1.8 (13.2) 384.7 ± 6.3 (45) 435.6 ± 8.28 (46) BrdU⁺ GFAP⁺ cells 0.3 ± 0.1 (0.6) 34.8 ± 2 (14.7) 205.2 ± 3.36 (24) 208.3 ± 3.96 (22) BrdU⁺ NeuN⁺ cells 0.1 ± 0.1 (0.3) 19.92 ± 1.2 (8) 102.6 ± 1.68 (12) 142 ± 2.7 (15) BrdU⁺ NF-L⁺ cells ― 0.7 ± 0.1 (0.3) 68.4 ± 1.12 (8) 94.7 ± 1.8 (10) Numbers in the parenthesis represent percentages (Numbers of cells / total BrdU⁺ cells).

6. CXCR4 and chemokine expression

To investigate whether application of hMSC promoted cell migration in the ischemic brain, I assessed SDF-1 levels in the brain (Fig. 6A). Tissue levels of SDF-1 were higher in the ischemic rat brains than in control brains (P<0.05). However, tissue levels of SDF-1 (Fig.

6A) and SDF-1 expression on immunostaining (data not shown) were not different between the tMCAo and hMSC-treated groups. In addition, flow cytometric analysis showed that both P2 and P6 hMSC stained for the CXCR4 antigens (Fig. 6B). However, surface expression of CXCR4 was less than 1% in both P2 and P6 hMSC, and no significant difference was found between them.

Fig. 6. Chemokine expression. (A) SDF-1 chemokine expression in the ischemic brain 14 days after tMCAo. (a) Sham group, (b) tMCAo-only, (c) tMCAo + P6 MSCs, and (d) tMCAo + P2 MSCs. No significant difference was found between the tMCAo-only group

7. Growth factor level

Using a sandwich ELISA, I examined tissue levels of VEGF, HGF, NGF, GDNF, BDNF, and bFGF in ischemic brain tissue 14 days after tMCAo. As shown in Fig. 7, the levels of trophic factors were significantly higher in the brain tissue of rats that received intravenous application of hMSCs than in those of the tMCAo group (P<0.05 in all cases). The brain levels of VEGF, HGF, NGF, and GDNF were significantly higher in the brain tissue of rats that received intravenous application of P2 hMSCs than in those of the P6-hMSC group (P<0.05 in all cases). The level of BDNF was similar between the P2 and P6 hMSCs group, whereas the level of bFGF was higher in the P6 hMSC group than in the P2 hMSC group.

Fig. 7. Brain levels of trophic factors at 14 days after tMCAo. (A) vascular endothelial growth factor (VEGF), (B) hepatocyte growth factor (HGF), (C) nerve growth factor (NGF), (D) glial cell line-derived neurotrophic factor (GDNF), (E) brain-derived neurotrophic factor (BDNF), and (F) basic fibroblast growth factor (bFGF). ^*P<0.05, ^**P<0.01, ^***P<0.001.

IV. DISCUSSION

My results are consistent with previous reports that functional recovery occurs after treatment with hMSCs. However, the mechanisms underlying the beneficial effects of these therapies have not been fully investigated. MSCs promote endogenous plasticity, angiogenesis, and neurogenesis (Chen et al., 2003; Zhang et al., 2003; Zhang et al., 2001).

The functional benefit of these cells is derived from either their ability to differentiate or integrate into cerebral tissue and act as stem or progenitor cells (Chen et al., 2001a; Chen et al., 2001b; Cogle et al., 2004; Deng et al., 2005; Pisati et al., 2007), spontaneous cell fusion (Terada et al., 2002; Ying et al., 2002), or from their ability to cause endogenous restorative activity by inducing production of neurotrophic factors (Chen et al., 2001a; Savitz et al., 2002; Wang et al., 2007; Zhao et al., 2006). My immunohistochemistry results indicate that most of the endogenous neuronal progenitor cells (BrdU-positive), but no hMSCs (NuMA-positive), expressed neuronal or glial phenotypes. Our data suggest that rather than transdifferentiation, up-regulation of the endogenous recovery mechanism at the peri-infarct area (neurogenesis) is an important role of hMSCs in functional recovery after ischemic stroke. This benefit might reflect the production of growth factors, including neurotrophins, which might promote the repair of damaged parenchymal cells, reduce apoptosis in the IBZ,

good agreement with a previous report that hMSCs have the capacity to secrete trophic factors in culture in response to ex vivo treatment with ischemic brain extract (Chen et al., 2002). The ability of MSCs to secrete multiple growth factors and cytokines suggests their important trophic roles in a plethora of cellular and physiological functions (Majumdar et al., 2000; Majumdar et al., 1998; Wieczorek et al., 2003).

Given that application of MSCs, trophic factors, or both, has been reported to be associated with reduced infarct size, the behavioral recovery observed in the present study may result solely from the neuroprotective effects of hMSCs against ischemic brain injury.

Recent experimental studies suggest that gene transduction into MSCs can enhance their therapeutic potential (Kurozumi et al., 2005; Lichtenwalner et al., 2006). Various trophic factor gene-modified MSCs were reported to be associated with improved behavioral recovery in a stroke model, including GDNF (Horita et al., 2006), HGF (Zhao et al., 2006), BDNF (Noumura et al., 2005), and placental growth factor (a VEGF family member) (Liu et al., 2006). Application of VEGF over-expressing MSCs has also been reported to reduce infarct size in a myocardial infarction model (Matsumoto et al., 2005).

In the present study, although the hMSC-treated groups showed improved functional recovery compared to the control group, no significant difference in infarct size was observed between the hMSC-treated and control group. Possible explanations include the following. First, I used naive MSCs, and thus the degree of expression of trophic factor proteins in my hMSC groups may be lower than those of gene-modified MSCs. Second, in the present study, the time of intravenous application of hMSCs was 24 h after tMCAo,

In the present study, although the hMSC-treated groups showed improved functional recovery compared to the control group, no significant difference in infarct size was observed between the hMSC-treated and control group. Possible explanations include the following. First, I used naive MSCs, and thus the degree of expression of trophic factor proteins in my hMSC groups may be lower than those of gene-modified MSCs. Second, in the present study, the time of intravenous application of hMSCs was 24 h after tMCAo,