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Analysis of Therapeutic Effect after Human Mesenchymal Stem Cell Transplantation in Ischemic Stroke Rat

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Analysis of Therapeutic Effect after

Human Mesenchymal Stem Cell

Transplantation in Ischemic Stroke Rat

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Mesenchymal Stem Cell Transplantation in

Ischemic Stroke Rat

by

Wen Yu Li

A Dissertation Submitted to The Graduate School of Ajou University

In Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Supervised by

Gwang Lee, Ph.D.

Department of Medical Sciences

The Graduate School, Ajou University

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李文玉의 醫學 博士學位 論文을 認准함.

審査委員長 안 영 환 印

審 査 委 員 이 광 印

審 査 委 員 주 인 수 印

審 査 委 員 이 필 휴 印

審 査 委 員 박 중 진 印

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I would like to express my sincere gratitude to all of you who in various ways

helped me to complete this thesis.

Thanks to professor Gwang Lee, Oh Young Bang, Young Hwan Ahn, Phil Hyu

Lee, and Neuroregeneration and Stem Cell Research Center member of Dr.

Man Jeong Paik, and all of students (박현정, 심우영, 신진영, 김유정, 문승민,

조은영)in Neuroregeneration and Stem Cell Research Lab. at Ajou University.

Also, thanks to professor In Soo Joo, director of department of Neurology, Ajou

University. Thanks to professor Mei Zi Jiang, colleague Yong Min Jin,

Department of Neurology, Yan Bian University in China.

Special thanks to my parents, my father and mother, act such as compass of life

to me. Also, thanks to my parents-in-law and sisters-in-law (Sheng Hua Jin and

Sheng xi Jin), always love me and support me.

My cousin's sister Ming Shu Zheng, for giving me invaluable support.

As the last mentions, I would like to give this thesis to my honey husband Ri

Long, if haven’t your love, I can not do all this.

“Never give up on what you really want to do.

The person with big dreams is more powerful than one with all facts. ”

2007. 12. 21

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Analysis of Therapeutic Effect after Human Mesenchymal Stem Cell

Transplantation in Ischemic Stroke Rat

Background and Purpose:

Mesenchymal stem cells (MSCs) have recently been investigated as an attractive therapeutic

tool for ischemic stroke because of their plasticity and availability. For the understanding of therapeutic effect of MSCs after transplantation in ischemic rats, changes in free fatty acids

(FFA) levels and proteins were detected in 4 days after MSC transplantation (Part 1, 2). I also evaluated the impact of the passage of MSCs on their effects in a rat stroke model (Part 3).

Part I: Although ex vivo culture-expansion is necessary to use autologous MSCs in treating

stroke patients and several researchers have utilized culture-expanded cells in their studies,

the effects of culture-expansion on neurogenesis and trophic support are unknown. Thus, I evaluated the impact of the passage of MSCs on their effects in a rat stroke model. The

intravenous application of ex vivo-cultured human MSCs, earlier (passage 2) or later passage (passage 6), was performed in a rat stroke model. Compared to rats that received

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in earlier-passage MSC-treated brains than in control or later-passage MSC-treated brains (P<0.01 in all cases). This study indicated that ischemia-induced neurogenesis was enhanced

by the intravenous administration of human MSCs. The effects were more pronounced with earlier-passage than with later-passage human MSCs, which may be related to the

differential capacity in trophic support, depending on their passage.

Part II: For the understanding of the complexity of biochemical and physiological changes

of hMSC-treated MCAo rat, I performed proteomic analysis of sham (n=3), tMCAo-only group (n=3) and hMSC-treated tMCAo rat group (n=3). Using 2-dimensinal electrophoresis

(2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), I was able to identify 14 proteins. Among the 14 identified proteins, 11 proteins were up-regulated or down-regulated in the hMSC-treated group compared to the tMCAo group, three proteins recovered to their normal condition after hMSC treatment. Differential protein expression was confirmed by western blotting. Up-regulated proteins such as Annexin A3 and GRP78 are involved in angiogenesis and neuroprotection. Recovered proteins such as synaptosomal-associated protein 25(SNAP-25) and transitional endoplasmic reticulum ATPase are involved in neuron loss and apoptosis. This study

established differential proteomic profiles that characterize hMSC transplanted MCAo rat. The proteomic profiles helped to explain the MSC action mechanism of stroke therapy.

Part III: Mesenchymal stem cells (MSCs) have the potential to promote brain repair and

improve recovery following stroke. I investigated changes in FFAs following intravenous

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transplantation of hMSC or phosphate-buffered saline (PBS) at one day after MCAo. All rats were sacrificed 5 days after MCAo. Metabolic profiling of free fatty acids (FFAs) levels was

assessed in plasma and brain from control rats (n=8), PBS-treated MCAo rats (n=6), and hMSC-treated MCAo rats (MCAo + hMSC, n=6). The levels of some FFAs in plasma and

brain samples of the MCAo and MCAo + hMSC groups were significantly different than those of the control group. The percentage composition of myristic acid in plasma, and of

myristic acid, linoleic acid, and eicosenoic acid in brain tissues of the MCAo + hMSC group were significantly reduced compared to those in the untransplanted MCAo group. My

metabolic approach has provided insights into understanding the events that occur in ischemic brain injury and the therapeutic effects of MSCs in stroke. This approach may be

useful to monitor the therapeutic effects of hMSC transplantation in the rat cerebral ischemia model.

Key words: Ischemic stroke; Mesenchymal stem cells; Neurogenesis; Passage; 2-dimensinal

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z PART I

ABSTRACT --- i

TABLE OF CONTENTS --- iv

LIST OF FIGURES --- vii

LIST OF TABLES --- ix

I. INTRODUCTION --- 1

II. MATERIALS AND METHODS --- 3

1. Animal Model --- 3 2. Experimental Groups --- 3 3. Human MSC Culture --- 4 4. MTT Assay --- 5 5. Flow Cytometry --- 5 6. Functional Tests --- 6

7. MRI Studies and Measurement of Infarct Volume --- 6

8. Histological and Immunohistochemical Assessment --- 7

8. 1. TTC Staining and Quantitative Analysis of Infarct Volume --- 7

8. 2. Tissue Preparation and Immunohistochemistry --- 8

8. 3. Stereological Cell Counts --- 9

8. 4. Immunofluorescence Assays --- 10

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III. RESULTS --- 13 IV. DISCUSSION --- 25 V. CONCLUSION --- 33 REFERENCES --- 34

z PART II

I. INTRODUCTION --- 44

II. MATERIALS AND METHODS --- 46

1. Transient MCAo Animal Model --- 46

2. Experimental Groups --- 46

3. Human MSC Culture --- 47

4. Preparation of Rat Brain Tissues Protein Samples --- 47

5. Two-dimensional Electrophoresis (2-DE) --- 48

6. Protein Visualization and Image Analysis --- 49

7. In-gel Digestion --- 49

8. MALDI-TOF-MS and Database Search --- 50

9. Western Blot Analysis --- 51

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I. INTRODUCTION --- 74

II. MATERIALS AND METHODS --- 77

1. Transient MCAo Animal Model --- 77

2. Experimental Groups --- 77

3. Human MSC Culture --- 78

4. 2, 3, 5-Triphenyltetrazolium Chloride (TTC) Staining --- 80

5. Immunohistochemistry --- 80

6. Chemicals and Reagents --- 81

7. Preparation of Plasma Samples --- 81

8. Preparation of Brain Samples --- 82

9. Gas Chromatography-Mass Spectrometry --- 82

10. Star Pattern Recognition Analysis Using Plasma and Brain Samples ---- 83

III. RESULTS --- 84

IV. DISCUSSION --- 92

V. CONCLUSION --- 96

REFFERENCES --- 97

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z PART I

Fig. 1. Characteristics of MSC --- 14 Fig. 2. Results of behavioral tests and infarct volume measurements --- 16 Fig. 3. Comparison of neurogenesis between the groups --- 19 Fig. 4. Phenotype of NuMA-labeled cells in the boundary zone 14 days after tMCAo

--- 20

Fig. 5. Phenotype of BrdU-labeled cells in the subventricular and boundary zone 14 days after tMCAo --- 21 Fig. 6. Chemokine expression --- 23 Fig. 7. Brain levels of trophic factors at 14 days after tMCAo --- 24

z PART II

Fig. 1. Overview 2-DE maps of rat brain tissue --- 55 Fig. 2. Distinguishing proteins on the cropped gel images --- 58 Fig. 3. MALDI-TOF mass spectra of tryptic digests of two protein spots resolved on

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Fig. 1. Flow cytometric analysis of hMSC --- 79

Fig. 2. TTC and hMSC staining --- 85

Fig. 3. Using star symbol plats overview of serum metabolism changes --- 88

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z PART I

Table 1. Quantitative analysis of BrdU-labeled cells and their phenotypes --- 22

z PART II

Table 1.

Spot matching summary ---

53

Table 2. List of identified proteins with significantly different expression among three groups --- 56

z PART III

Table 1. The percentage composition of 24 FFAs found in rat plasma --- 87 Table 2. The percentage composition of 24 FFAs found in brain tissues --- 90

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PART I

What we really lose with ex

vivo-cultivation of mesenchymal stem cells:

roles in induction of endogenous

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I. INTRODUCTION

The use of mesenchymal stem cells (MSCs) as a therapy for stroke is attractive because autologous MSCs could be used, which would avoid immune reactions and ethical issues.

The multi-lineage potential of MSCs has been under intense scrutiny in recent years due to the documented success in using transplantation to treat human disorders (Strauer et al.,

2002; Tateishi-Yuyama et al., 2002; Tse et al., 2003). Transplantation of bone marrow stromal cells into animal models of cerebral ischemia has been shown to reduce lesion size

and improve functional outcome (Chen et al., 2001a; Chen et al., 2001b; Kurozumi et al., 2004; Yasuhara et al., 2006).

From a cell therapy perspective, ex vivo culture-expansion of MSCs before transplantation is necessary to meet the dose requirements that are effective in animal models

because relatively few MSCs can be obtained by bone marrow aspiration. Recent advances in the capacity for large-scale expansion of MSCs have led to clinical trials to assess the

safety and efficacy of MSC transplantation for a variety of pathological conditions.

We have previously reported the results of a clinical trial using autologous MSCs in patients

with severe ischemic stroke (Bang et al., 2005). Our preliminary results demonstrated the feasibility and safety in administering ex vivo-cultured autologous MSCs intravenously to

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in those patients (Savitz et al., 2002).

While many researchers have used culture-expanded cells in their studies, the effects of culture expansion on neurogenesis and trophic support remain unknown. Thus, I investigated

the impact of ex vivo culturing of human MSCs (hMSCs) on neurogenesis and trophic support in a rat model of ischemic stroke. I also evaluated possible mechanisms of the effects

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II. MATERIALS AND METHODS

1. Animal Model

The use of animals in this study was approved by the Animal Care and Use Committee of Ajou University, and all procedures were carried out in accordance with institutional

guidelines. Male Sprague–Dawley rats (250–300 g) were anesthetized with 4% isoflurane and maintained with 1.5% isoflurane in 70% N2O and 30% O2 using a face mask. Rectal

temperature was maintained at 37.0–37.5°C with heating pads. Transient MCAo was induced using a method of intraluminal vascular occlusion that has been modified in our

laboratory (Chen et al., 1992; Wang et al., 2007). A 4–0 surgical monofilament nylon suture with a rounded tip was moved from the left common carotid artery into the lumen of the

internal carotid artery to block the origin of the MCAo. Two hours after MCAo, reperfusion was performed by the withdrawal of the suture to the tip of the common carotid artery.

2. Experimental Groups

One day after tMCAo, the animals were randomly divided into four groups (n = 7 for each group): sham operation + phosphate-buffered saline (PBS) injection, tMCAo + PBS

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dose of MSCs (~1 × 108 cells/patient), equivalent to the doses shown to be effective in the rat

model of stroke (Bang et al., 2005). Animals were not immunosuppressed after hMSC transplantation.

All animals received daily intraperitoneal (i.p.) injections of 50 mg/kg bromodeoxyuridine (BrdU, a thymidine analog; Roche, Nutley, NJ), which labels newly

synthesized DNA (27), starting 24 h after tMCAo for 13 consecutive days. All animals were killed with 10% chloral hydrate 14 days after tMCAo.

3. Human MSC Culture

hMSCs were obtained from 20-mL aspirates from the iliac crest (Bang et al., 2005; Chen et al., 2003; Li et al., 2002). Each 20-mL aspirate was diluted 1:1 with PBS and layered over

10 mL of Ficoll (Ficoll-Paque; Amersham Biosciences, Piscataway, NJ). After centrifugation (2,000 rpm, 20 min), the mononuclear cell layer was removed from the interface and

suspended in PBS. Cells were again centrifuged (1,200 rpm, 5 min) and re-suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum.

The cells were incubated at 37°C in 5% CO2 in flasks for 1 day and non-adherent cells were

removed by replacing the medium. After the cultures reached confluence, usually within 1 to

2 weeks, the cells were harvested with 0.05% trypsin and 0.53 mmol/L EDTA (GIBCO, Gaithersburg, MD) for 5 min at 37°C, replated in a flask, cultured for another 1 week, and

harvested. The cells used in these experiments were harvested at P2 and P6. The protocol was approved by the local institutional review board.

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4. MTT Assay

hMSCs from passages P2, P6, and P15 were plated onto 24-well plates (104 cells/well) in

DMEM + 10% FBS. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]

assays were performed 1, 2, 3, and 4 days after plating, using an established protocol (Chen et al., 1998). MTT (0.25 mg/mL) was added to each well and cells were incubated at 37°C

for 20 min, after which the medium was replaced by 0.2 mL DMSO per well. MTT reduction was determined by measuring the OD540nm of the DMSO extracts using DMSO as blank.

5. 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

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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

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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

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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

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hemisphere, every 6th 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).

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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

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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

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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.

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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).

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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.

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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

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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

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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.

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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

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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

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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,

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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

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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).

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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)

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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.

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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,

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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,

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intravenous administration of bone marrow in reducing lesion size after cerebral ischemia

has been reported (Iihoshi et al., 2004). Transplanted MSCs in the tMCAo model 24 h after ischemia occurred had improved functional recovery, but did not significantly decrease the

infarct area (Chen et al., 2001; Li et al., 2002).

My results suggest that the difference in functional recovery between the groups may

have resulted from the difference in the levels of trophic factors in brain tissue and also in the degree of neurogenesis. In most previous studies, only neuroprotective effects, in terms

of infarct size, were analyzed (Horita et al., 2006; Liu et al., 2006; Nomura et al., 2005; Zhao et al., 2006); other aspects of therapeutic benefit, such as neurogenesis, have seldom been

reported. Given the relatively few cells showing brain cell phenotype and the absence of marked reduction in infarct size, neither neurogenesis nor neuroprotection may entirely

account for the behavioral recovery of the hMSC-treated rats.

My results showed that ischemia-induced neurogenesis was enhanced by the application

of hMSCs. Further studies are needed to evaluate whether the increased BrdU-positive cells are the result of increased proliferation, enhanced survival in a toxic environment, or both.

Intraventricular infusion of VEGF increased neurogenesis, but it is unclear whether this effect resulted from increased proliferation, survival, or both (Lichtenwalner et al., 2006).

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provide various cytokines and trophic factors, which may be important in repair mechanisms

after stroke (Prockop et al., 1997). Cell transplantation may provoke diverse mechanisms involved in the structural modifications of dendrites and synapses associated with function

plasticity after ischemic injury (Dunnett et al., 1994; Iihoshi et al., 2004). Moreover, application of bone marrow stromal cells has recently been reported to reduce scar thickness

and increase the number of oligodendrocyte precursor cells (Shen et al., 2007). It was suggested that functional recovery may be enhanced by the interaction between MSCs and

the host brain in an anatomically distributed, tissue-sensitive, and temporally ongoing way (Zhao et al., 2006).

In the present study, the therapeutic benefit of MSC transplantation was greatly influenced by the passage of the hMSCs; the application of earlier-passage hMSCs showed

more therapeutic benefit than the later-passage cells. Concerning the characteristics and potentiality of MSCs, my results about the effect of culture expansion on stem cell

potentiality in neurogenesis and trophic support may have important implications in both clinical trials involving cell therapy and basic research. My data demonstrate that

intravenous hMSC treatment promotes neurogenesis in the SVZ and IBZ and that this effect is more pronounced with earlier-passage than with later-passage MSCs. Recently, it was

reported that the ability to differentiate into neurons, osteocytes, and adipocyte was more effective in P2 rat MSCs than in P10 cells (Yeon et al., 2006), and several studies have

examined the growth pattern of MSCs (Liu et al., 2003; Zhang et al., 2005). However, to my knowledge, no report has compared the effects of MSCs on neurogenesis after stroke,

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Various trophic factors influence neurogenesis (proliferation, survival, and

differentiation) in the mature brain (LIchtenwalner et al., 2006), and the capacity to release trophic factors has been suggested as key to the beneficial effect of MSCs in cerebral

ischemia (Chen et al., 2002). The application of gene-modified hMSCs is an attractive modality to increase trophic support by hMSCs. However, this method is not feasible in

clinical practice.

I hypothesized that characteristics of the hMSCs (such as passage number) would be

important when considering hMSCs for enhancing ischemia-induced neurogenesis and trophic support. To My knowledge, differences in the brain levels of trophic factors after

application of different-passage MSCs has not been previously reported. My data showed that neurogenesis was enhanced by the application of hMSCs and that the effects were more

prominent with earlier-passage hMSCs. Thus, it is conceivable that in the clinical application of hMSCs, a better result might be achieved by applying a smaller dose of earlier-passage

hMSCs, rather than waiting to achieve a higher number of later-passage hMSCs by further ex

vivo culturing.

In the present study, I examined possible reasons for the different capacities of earlier and later-passage hMSCs. Cell viability, the expression levels of chemokine SDF-1 (also

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analysis: when I treated cultured hMSCs ex vivo with ischemic brain extract, the levels of

trophic factors secreted by P2 hMSCs were higher than by P6 hMSCs (Choi et al., 2006). Levels of HGF, VEGF, and NGF markedly increased in cultured P2 hMSCs versus P6

hMSCs after ex vivo treatment with ischemic brain extract, whereas the BDNF level significantly increased in the latter versus the former (Choi et al., 2006).

Both our in vivo and in vitro data suggest that P2 and P6 hMSCs differ in their capacity for trophic support, resulting in superior efficacy of P2 hMSCs in neurogenesis compared to

P6 hMSCs. Several trophic factors have been reported to improve adult neurogenesis in ischemic-injured brain, including VEGF, epidermal growth factor, heparin-binding

epidermal growth factor, insulin-like growth factor, BDNF, GDNF, and bFGF (Emsley et al., 2005; Kobayashi et al., 2006; Teramoto et al., 2003; Wang et al., 2007; yasuhara et al.,

2004). Among these, the increased tissue level of VEGF in ischemic brain tissue was reported to be associated with neurogenesis (Emsley et al., 2005; Kobayashi et al., 2006;

Teramoto et al., 2003; Wang et al., 2007; yasuhara et al., 2004), as was observed in rats that received the earlier-passage hMSCs in the present study. The roles of GDNF, NGF, and

HGF in neurogenesis after ischemic injury remain unclear. Further studies using antagonists of these trophic factors are needed.

This study has some limitations. First, I compared only the differential effect of P2 and P6 hMSCs on neurogenesis; hMSCs of later passages were not evaluated. However, my

electron microscopy study showed that unlike later-passage hMSCs (such as P15) that show dieing cells, P2 and P6 hMSCs had similar morphologic features and the viability of P6

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apoptotic cell death and angiogenesis was not evaluated in the present study. It has been

suggested that the beneficial effects of MSCs may not result from neurogenesis; neuroprotective and angiogenetic effects may actually be primarily responsible (Chen et al.,

2001a). The differences in the number of BrdU-positive cells between the groups may have been caused by differences in the protective effects of neurotrophic factors against apoptotic

death of BrdU-positive cells as well as by enhanced neurogenesis per se. In addition, there were no differences in the in vitro survival between P2 and P6 in the present study.

However, in vivo survival rates of early and late passage hMSCs were not evaluated in this study; which could be related to the differential capacity in neurogenesis and trophic

supports between P2 and P6 hMSCs. Further studies are needed concerning the presence and degree of apoptotic cell death of hMSCs and BrdU-positive cells. Third, MSCs can give rise

to microglia, which in turn may play a major role in the response to ischemia. However, in the present study, I focused on the effect of the passage of the MSCs on neurogenesis and

trophic support, and the effects of hMSCs on microglia were not examined. Further studies concerning the immune-modulating effects of hMSCs are needed. Lastly, the numbers of

trophic factors involved in neurogenesis are increasing, but the tissue levels of all these trophic factors could not be tested in the present study. Recently, it was reported that VEGF

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animals infused with a lower dose of MSCs (1 × 106) (Chen et al., 2001a). Additional studies

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V. CONCLUSION

I demonstrated that hMSCs can enhance neurogenesis in the ischemic rat brain and that

this potential depends on their passage. Given that the neurogenic potential and trophic support of hMSCs differ depending on their passage, these aspects should be considered

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REFERENCES

1. Bang, O. Y.; Lee, J. S.; Lee, P. H.; Lee, G. Autologous mesenchymal stem cell

transplantation in stroke patients. Ann Neurol 57(6):874-882; 2005

2. Chen, H.; Chopp, M.; Zhang, Z. G.; Garcia, J. H. The effect of hypothermia on transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 12(4):621-628;

1992

3. Chen, J.; Li, Y.; Wang, L.; Lu, M.; Zhang, X.; Chopp, M. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats.

J Neurol Sci 189(1-2):49-57; 2001a

4. Chen, J.; Li, Y.; Wang, L.; Zhang, Z.; Lu, D.; Lu, M.; Chopp, M. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats.

Stroke 32(4):1005-1011; 2001b

5. Chen, J.; Li, Y.; Zhang, R.; Katakowski, M.; Gautam, S. C.; Xu, Y.; Lu, M.; Zhang, Z.; Chopp, M. Combination therapy of stroke in rats with a nitric oxide donor and human

bone marrow stromal cells enhances angiogenesis and neurogenesis. Brain Res 1005(1-2):21-28; 2004

(50)

6. Chen, J.; Zhang, Z. G.; Li, Y.; Wang, L.; Xu, Y. X.; Gautam, S. C.; Lu, M.; Zhu, Z.;

Chopp, M. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats. Circ Res 92(6):692-699;

2003

7. Chen, S. J.; Bradley, M. E.; Lee, T. C. Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: modulation by calcium-regulated proteases and protein

kinases. Mol Cell Biochem 178(1-2):141-149; 1998

8. Chen, X.; Li, Y.; Wang, L.; Katakowski, M.; Zhang, L.; Chen, J.; Xu, Y.; Gautam, S. C.; Chopp, M. Ischemic rat brain extracts induce human marrow stromal cell growth factor

production. Neuropathology 22(4):275-279; 2002

9. Choi, Y.J.; Li, W. Y.; Bang, O. Y. Enhancing the efficacy of mesenchymal stem cells in ischemic stroke by ex vivo treatment with trophic factors. Abstract. the Society for

Neuroscience, Atlanta, 2006

(51)

11. Deng, J.; Petersen, B. E.; Steindler, D. A.; Jorgensen, M. L.; Laywell, E. D.

Mesenchymal Stem Cells Spontaneously Express Neural Proteins in Culture, and Are Neurogenic After Transplantation. Stem Cells 24(4): 1054-1056; 2006

12. Dunnett, S. B. Behavioural consequences of neural transplantation. J Neuro 242(1 Suppl

1):S43-53; 1994

13. Emsley, J. G.; Mitchell, B. D.; Kempermann, G.; Macklis, J. D. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog

Neurobiol 75(5):321-341; 2005

14. Horita, Y.; Honmou, O.; Harada, K.; Houkin, K.; Hamada, H.; Kocsis, J. D. Intravenous administration of glial cell line-derived neurotrophic factor gene-modified human

mesenchymal stem cells protects against injury in a cerebral ischemia model in the adult rat. J Neurosci Res 84(7):1495-1504; 2006

15. Iihoshi, S.; Honmou, O.; Houkin, K.; Hashi, K.; Kocsis, J. D. A therapeutic window for

intravenous administration of autologous bone marrow after cerebral ischemia in adult rats. Brain Res 1007(1-2):1-9; 2004

(52)

16. Kobayashi, T.; Ahlenius, H.; Thored, P.; Kobayashi, R.; Kokaia, Z.; Lindvall, O.

Intracerebral infusion of glial cell line-derived neurotrophic factor promotes striatal neurogenesis after stroke in adult rats. Stroke 37(9):2361-2367; 2006

17. Kokaia, Z.; Thored, P.; Arvidsson, A.; Lindvall, O. Regulation of stroke-induced

neurogenesis in adult brain--recent scientific progress. Cereb Cortex 16 Suppl 1:i162-167; 2006

18. Kurozumi, K.; Nakamura, K.; Tamiya, T.; Kawano, Y.; Ishii, K.; Kobune, M.; Hirai,

S.; Uchida, H.; Sasaki, K.; Ito, Y; Kato K; Honmou, O; Houkin, K; Date, I; Hamada, H. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in

the rat middle cerebral artery occlusion model. Mol Ther 11(1):96-104; 2005

19. Kurozumi, K.; Nakamura, K.; Tamiya, T.; Kawano, Y.; Kobune, M.; Hirai, S.; Uchida, H.; Sasaki, K.; Ito, Y.; Kato, K; Honmou, O; Houkin, K; Date, I; Hamade, H. BDNF

gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model. Mol Ther 9(2):189-197; 2004

(53)

21. Lichtenwalner, R. J.; Parent, J. M. Adult neurogenesis and the ischemic forebrain. J Cereb Blood Flow Metab 26(1):1-20; 2006

22. Liu, H.; Honmou, O.; Harada, K.; Nakamura, K.; Houkin, K.; Hamada, H.; Kocsis, J. D.

Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia. Brain 129(Pt 10):2734-2745; 2006

23. Liu, Y.; Song, J.; Liu, W.; Wan, Y.; Chen, X.; Hu, C. Growth and differentiation of rat

bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res 58(2):460-468; 2003

24. Majumdar, M. K.; Thiede, M. A.; Haynesworth, S. E.; Bruder, S. P.; Gerson, S. L.

Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and

osteogenic lineages. J Hematother Stem Cell Res 9(6):841-848; 2000

25. Majumdar, M. K.; Thiede, M. A.; Mosca, J. D.; Moorman, M.; Gerson, S. L. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells

(54)

26. Matsumoto, R.; Omura, T.; Yoshiyama, M.; Hayashi, T.; Inamoto, S.; Koh, K. R.; Ohta,

K.; Izumi, Y.; Nakamura, Y.; Akioka, K; Kitaura, Y; Takeuchi, K; Yoshiyama, J. Vascular endothelial growth factor-expressing mesenchymal stem cell transplantation

for the treatment of acute myocardial infarction. Arterioscler Thromb Vasc Biol 25(6):1168-1173; 2005

27. Miller, M. W.; Nowakowski, R. S. Use of bromodeoxyuridine-immunohistochemistry to

examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res 457(1):44-52; 1988

28. Nomura, T.; Honmou, O.; Harada, K.; Houkin, K.; Hamada, H.; Kocsis, J. D. I.V.

infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat. Neuroscience

136(1):161-169; 2005

29. Pisati F, Bossolasco P, Meregalli M, Cova L, Belicchi M, Gavina M, Marchesi C, Calzarossa C, Soligo D, lambertenghi-Deliliers G, Bresolin N, silani V, Torrente Y,

(55)

30. Plane, J. M.; Liu, R.; Wang, T. W.; Silverstein, F. S.; Parent, J. M. Neonatal

hypoxic-ischemic injury increases forebrain subventricular zone neurogenesis in the mouse. Neurobiol Dis 16(3):585-595; 2004

31. Prockop, D. J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science

276(5309):71-74; 1997

32. Savitz, S. I.; Rosenbaum, D. M.; Dinsmore, J. H.; Wechsler, L. R.; Caplan, L. R. Cell transplantation for stroke. Ann Neurol 52(3):266-275; 2002

33. Shen, L. H.; Li, Y.; Chen, J.; Zacharek, A.; Gao, Q.; Kapke, A.; Lu, M.; Raginski, K.;

Vanguri, P.; Smith, A; Chopp, M. Therapeutic benefit of bone marrow stromal cells administered 1 month after stroke. J Cereb Blood Flow Metab 27(1):6-13; 2007

34. Strauer, B. E.; Brehm, M.; Zeus, T.; Kostering, M.; Hernandez, A.; Sorg, R. V.; Kogler,

G.; Wernet, P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106(15):1913-1918; 2002

35. Tateishi-Yuyama, E.; Matsubara, H.; Murohara, T.; Ikeda, U.; Shintani, S.; Masaki, H.;

Amano, K.; Kishimoto, Y.; Yoshimoto, K.; Akashi, H; Shimada, K; Iwasaka, T; Imaizumi, T. Therapeutic angiogenesis for patients with limb ischaemia by autologous

(56)

transplantation of bone-marrow cells: a pilot study and a randomised controlled trial.

Lancet 360(9331):427-435; 2002

36. Terada, N.; Hamazaki, T.; Oka, M.; Hoki, M.; Mastalerz, D. M.; Nakano, Y.; Meyer, E. M.; Morel, L.; Petersen, B. E.; Scott, E. W. Bone marrow cells adopt the phenotype of

other cells by spontaneous cell fusion. Nature 416(6880):542-545; 2002

37. Teramoto, T.; Qiu, J.; Plumier, J. C.; Moskowitz, M. A. EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia. J Clin Invest

111(8):1125-1132; 2003

38. Tse, H. F.; Kwong, Y. L.; Chan, J. K.; Lo, G.; Ho, C. L.; Lau, C. P. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell

implantation. Lancet 361(9351):47-49; 2003

39. Wang, Y. Q.; Guo, X.; Qiu, M. H.; Feng, X. Y.; Sun, F. Y. VEGF overexpression enhances striatal neurogenesis in brain of adult rat after a transient middle cerebral artery

(57)

41. Wieczorek, G.; Steinhoff, C.; Schulz, R.; Scheller, M.; Vingron, M.; Ropers, H. H.; Nuber, U. A. Gene expression profile of mouse bone marrow stromal cells determined

by cDNA microarray analysis. Cell Tissue Res 311(2):227-237; 2003

42. Yasuhara T, matsukawa N, Yu G, Xu Li, Mays RW, Kovach J, Deans RJ, Hess DC, Carrll JE, Borlongan CV.Behavioral and histological characterization of

intrahippocampal grafts of human bone marrow-derived multipotent progenitor cells in neonatal rats with hypoxic-ischemic injury. Cell Transplant 15(3): 231-238; 2006

43. Yasuhara, T.; Shingo, T.; Date, I. The potential role of vascular endothelial growth factor

in the central nervous system. Reviews in the neurosciences 15(4):293-307; 2004.

44. Yeon Lim, J.; Jeun, S. S.; Lee, K. J.; Oh, J. H.; Kim, S. M.; Park, S. I.; Jeong, C. H.; Kang, S. G. Multiple stem cell traits of expanded rat bone marrow stromal cells. Exp

Neurol 199(2):416-426; 2006

45. Ying, Q. L.; Nichols, J.; Evans, E. P.; Smith, A. G. Changing potency by spontaneous fusion. Nature 416(6880):545-548; 2002

(58)

46. Zhang, F. B.; Li, L.; Fang, B.; Zhu, D. L.; Yang, H. T.; Gao, P. J. Passage-restricted

differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun 336(3):784-792; 2005

47. Zhang, R.; Wang, L.; Zhang, L.; Chen, J.; Zhu, Z.; Zhang, Z.; Chopp, M. Nitric oxide

enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat. Circ Res 92(3):308-313; 2003

48. Zhang, R.; Zhang, L.; Zhang, Z.; Wang, Y.; Lu, M.; Lapointe, M.; Chopp, M. A nitric

oxide donor induces neurogenesis and reduces functional deficits after stroke in rats. Ann Neurol 50(5):602-611; 2001

49. Zhao, M. Z.; Nonoguchi, N.; Ikeda, N.; Watanabe, T.; Furutama, D.; Miyazawa, D.;

Funakoshi, H.; Kajimoto, Y.; Nakamura, T.; Dezawa, M; Shibata, M. A; Otsuki, Y; Coffin, R. S; Liu, W. D. Novel therapeutic strategy for stroke in rats by bone marrow

stromal cells and ex vivo HGF gene transfer with HSV-1 vector. J Cereb Blood Flow Metab 26(9):1176-1188; 2006

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PART II

Proteomic Analysis of Ischemic Rat

Brain after Human Mesenchymal Stem

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Bone marrow derived mesenchymal stem cells (MSC) can be differentiated into neurons,

and are considered to be a promising source of the treatment of neurological disease, such as stroke (Chopp and Li, 2002), Parkinson`s disease (Dezawa et al., 2004), and

muscle-degenerative disease (Dezawa et al., 2005). Notably, stroke is the third most common cause of death after cancer. Destruction of neurons caused by cerebral infarction eventually leads

to fatal damage in the function of the brain.

Cell replacement therapy has potential in biological replacement cells for the damaged

structure. Amongst them, human MSC can be implemented auto-graft therapy, and clinical research was reported (Bang et al., 2005). In addition, transplantation of human bone

marrow-derived stromal cells into animal models of cerebral ischemia has been shown to reduce lesion size, improve functional outcome, and induce neurogenesis (Kurozumi et al.,

2004; Li et al., 2002; Li et al., 2001).

MSCs were able to delay cell death and involved in restoring tissues through transient

trophic effect or cytokine release (Borlongan et al., 2004; Chopp and Li, 2002; Chopp et al., 2000; Nomura et al., 2005). However, the mechanism at the molecular level is still unknown.

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occlusion (tMCAo) group and hMSC-treated tMCAo group. I used 2-DE for observing the expression level of protein of the three groups, while setting the objective of this experiment

in the identification of different protein expression. I have also made 2-DE maps and database for the protein expression that occurs the during hMSC-treated tMCAo rat in this

experiment for the first time. This protein expression patterns could be used to the elucidation of hMSC therapy effect at a molecular level.

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1. Transient MCAo Animal Model

The use of animals in this study was approved by the Animal Care and Use Committee of Ajou University, and all procedures were carried out in accordance with institutional

guidelines. Male Sprague–Dawley rats (250–300 g) were anesthetized with 4% isoflurane and maintained with 1.5% isoflurane in 70% N2O and 30% O2 using a face mask. Rectal

temperature was maintained at 37.0–37.5°C with heating pads. Transient MCAo was induced using a method of intraluminal vascular occlusion that has been modified in our laboratory

(Chen et al., 1992). A 4–0 surgical monofilament nylon suture with a rounded tip was moved from the left common carotid artery into the lumen of the internal carotid artery to block the

origin of the MCAo. Two hours after MCAo, reperfusion was performed by the withdrawal of the suture to the tip of the common carotid artery.

2. Experimental Groups

One day post MCAo or sham, animals were divided into three groups: sham operation +

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hMSCs were obtained from 20 mL aspirates from the iliac crest of normal human donors (Bang et al., 2005) as part of a protocol approved by the Scientific-Ethical Review Board of

Ajou University Medical Center. Briefly, cells were incubated in 150 cm2 rectangular canted neck cell culture flasks (Corning Incorporated Life Sciences, USA) at 37°C in 5% CO2 for 1

day and non-adherent cells were removed by replacing the medium. The medium was then changed every 2 to 3 days until the adherent cells became 80% confluent. The cells were

harvested with 0.05% trypsin and 0.53 mmol/L EDTA (Gibco) for 5 min at 37°C, replated in a flask, and cultured for an additional 3 to 5 days, before they were harvested. Cells used in

these experiments were harvested after six passages. The expression levels of the MSC surface markers CD105 and CD73 in hMSC were evaluated using flow cytometry (n=3;

FACScan; Becton-Dickinson, Rurtherford, NJ). MSCs showed high levels of expression of stem cell markers CD105 and CD73 (data not shown).

4. Preparation of Rat Brain Tissues Samples

Frozen ischemia brain hemisphere tissues were homogenized within a detergent lysis buffer containing 7M urea, 2 M thiourea, 4% (w/v) Chaps, 0.5% (v/v) Triton X-100, 0.5%

(v/v) Pharmalytes pH 4-7(Amersham Biosciences, NJ), 100 mM DTT, and 1.5 mg/mL complete Protease Inhibitor Cocktail for mammalian tissues (Sigma–Aldrich, MO),

sonicated, and incubated for 1 h at room temperature in an orbital shaker. The lysate was then centrifuged at 13,000 rpm for 30 min. The total protein concentration of each sample

(64)

5. Two-dimensional Electrophoresis (2-DE)

For isoelectric focussing (IEF), IPG strips were used according to (Gorg et al., 2000) the supplier’s instructions. 200 μg of total proteins was mixed in a rehydration buffer (7 M urea,

2M thiourea, 2% Chaps, 0.5% Triton X-100, 100 mM DTT, 0.6% Pharmalytes pH 4-7, and bromophenol blue) in total volume of 340 μL and loaded on 18 cm pH 4-7 NL Immobiline

DryStrip (an IPG strip, Amersham biosciences, NJ). After IPG strip rehydration,IEF was done initially at 250 V for 15 min, and then the voltage was increased to 10,000 V within 3 h,

and maintained at 10,000 V for 7 h All IEF steps were carried out at 20℃ using pHaser Isoelectric Focusing System (Genomic Solutions, MI). After the first-dimensional IEF, IPG

gel strips were placed in an equilibration solution (6 M urea,2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) containing 1% DTT for 10 min with shaking at 50 rpm on an orbital

shaker. The gels were then transferred to the equilibration solution containing 2.5% iodoacetamide and shaken for a further 10 min before placing them on a 7.5% - 17.5%

gradient polyacrylamide gel slab (20x 20cm).Separation in the second dimension was carried out using Protean II xi electrophoresis equipment and Tris-glycine buffer (25 mM Tris, 192

(65)

For silver staining, following second-dimensional SDS-PAGE, analytical gels were immersed in methanol: acetic acid: water (50:12:38) for 1.5 h, followed by washing twice in

50% ethanol for 20 min. Gels were pretreated for 1 min in a solution of 0.02% Na2S2O3. This

was followed by three 1 min washes in deionized water. Proteins were stained in a solution

containing 0.2% AgNO3 and 0.075% v/v formalin (37% formaldehyde in water) for 20 min,

and washed twice in deionized water for 1 min. Subsequently, gels were developed in a

solution of 0.06% v/v formalin, 2% Na2CO3, and 0.0004% Na2S2O3. When the desired

intensity was attained, the developer was discarded and stopped by 1% acetic acid. Gel

image matching was done with PDQuest software (Version 7.3; Bio-Rad). Scanned gel images were processed to remove backgrounds, staining on the gel borders and to

automatically detect spots. For all spot intensity calculations, normalized values were used. Normalization of spot intensity was done so that the total sum of intensities in a gel would be

equal to 1,000,000, and normalized spot intensities were expressed in ppm.

7. In-gel Digestion

In-gel digestion of protein spots on silver stained gels was performed essentially as

described (Jensen et al., 1999). After the completion of staining, the gel slab was washed twice with water for 10 min. The spots of interest were excised with a scalpel, cut into pieces,

and put into 1.5 mL microtubes. The particles were washed twice with water for 15 min, and then twice with water/acetonitrile (1:1 v/v) for 15 min. The solvent volumes were about

(66)

NH4HCO3 for 5 min. Acetonitrile was added to give a 1:1 v/v mixture of 0.1 M

NH4HCO3/acetonitrile and the mixture was incubated for 15 min. All liquid was removed

and gel particles were dried in a vacuum centrifuge (Heto-Holten, Allerød, Denmark), reswelled in 10 mM DTT/0.1 M NH4HCO3, and incubated for 45 min at 56 ℃ to reduce the

peptides. After chilling tubes to room temperature and removing the liquid, 55 mM iodoacetamide in 0.1 M NH4HCO3 was added, the tubes were incubated for 30 min at room

temperature in the dark to S-alkylate the peptides. Iodoacetamide solution was removed, the gel particles were washed with 0.1 M NH4HCO3 and acetonitrile, dried in a vacuum

centrifuge, rehydrated on ice in digestion buffer containing 50 mM NH4HCO3, 5 mM CaCl2,

and 12.5 ng /μL of trypsin, and incubated for 45 min on ice. Excess liquid was removed and about 20 μL of digestion buffer without trypsin was added. After overnight digestion at

37 ℃, 25 mM NH4HCO3 was added, and the tube was incubated for 15 min. Acetonitrile

was added and the tube was incubated for a further 15 min. The supernatant was recovered, and the extraction was repeated twice with 5% formic acid/acetonitrile (1:1 v/v). The three

extracts were pooled and dried in a vacuum centrifuge.

수치

Fig. 1.    Characteristics of MSC    --------------------------------------------------------------  14  Fig
Table 1.    Quantitative analysis of BrdU-labeled cells and their phenotypes   --------  22
Fig. 1. Characteristics of MSCs. (A) Viability of P2, P6, and P15 hMSCs. Cells were
Fig. 2. Results of behavioral tests and infarct volume measurements. Adhesive-removal
+7

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