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Development of Combinatorial Strategies Employing Neural Stem Cells and Polymer Scaffold for Spinal Cord Repair

DC Field Value Language
dc.contributor.author강, 영미-
dc.date.accessioned2011-04-13T05:28:05Z-
dc.date.available2011-04-13T05:28:05Z-
dc.date.issued2009-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/2265-
dc.description.abstract"ABSTRACT-



Development of Combinatorial Strategies Employing Neural Stem Cells and Polymer Scaffold for Spinal Cord Repair



Stem cell-based therapy holds promise to enhance functional recovery following spinal cord injury (SCI). Most evidence that transplantation of neural stem cells can produce beneficial outcomes after SCI has been derived from rodent models. Before being translated to human patients, it would be needed to examine the therapeutic effects in larger animal models where weight-bearing locomotion should be much more challenging. The present study examined therapeutic effects of a combinatorial strategy centering on human neural stem cells (hNSCs) in a canine hemisection SCI model. After traumatic injuries to the spinal cord, secondary injuries aggravate the extent of damage, expanding cystic cavities at the lesion site. Cavity wall is surrounded by glial scar and severely impedes axonal regrowth. Therefore, any regenerative strategy could be complemented with an attempt to bridge lesion cavities. In the current study, we implanted poly (lactic-co-glycolic acid) (PLGA) scaffolds to facilitate the delivery of neural stem cells. We sought to enhance therapeutic efficacy by ectopic expression of neurotrophin-3 (NT-3) in the hNSCs. NT-3 overexpressing hNSCs (NT3.F3) were produced by retroviral transduction of the immortalized hNSC line (F3) with human NT-3 cDNA. NT3.F3 cells were seeded into a predesigned PLGA (65:35) scaffolds, and the PLGA scaffolds with hNSCs (PLGA-NT3.F3) were implanted immediately after left hemisection at T11 in female dogs weighing 20-30 kg. The PLGA scaffold seemed to nicely fill the lesion cavity, showing a varying degree of biodegradation by 12 weeks. Survival of grafted cells was confirmed at 2 weeks, and some of the grafted cells migrated to the host tissue. There were very few regenerating axons into the scaffold in both groups. Moreover, the ventral horns caudal to the hemisected region were more profusely innervated by serotonergic axons in animals with PLGA-NT3.F3. These findings raise a possibility that implantation of PLGA-NT3.F3 can produce beneficial motor outcomes by promoting axonal remodeling below the lesion. This study suggests that the therapeutic strategy combining multidisciplinary approaches can be feasible and effective for spinal cord repair in larger species.







Key words: spinal cord injury; neural stem cells; NT3; PLGA scaffold; serotonergic axon





















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dc.description.tableofcontents"TABLE OF CONTENTS



ABSTRACT i

TABLE OF CONTENTS iii

LIST OF FIGURES v LIST OF ABBREVIATION vi

I. INTRODUCTION 1

II. MATERIALS AND METHODS 5

1. Culture of immortalized human neural stem cells (hNSCs) 5

2. Pretreatment of scaffold and cell seeding 5

3. ELISA of NT-3 6

4. Spinal cord injury and implantation of scaffold 7

5. Histological preparation . 8

6. Immuohistochemistory 8

7. Quantitative morphometry9

III. RESULTS12

1. Seeding NT-3 overexpressing hNSCs in PLGA scaffold 12

2. Implantation of PLGA seeded with hNSCs (PLGA-NT3.F3) 15

3. Detection of human neural stem cell in scaffold 18

4.Deposition of chondroitin sulfate proteoglycans (CSPG) in the lesion 22

5. Remodeling of serotonergic axons 24

IV. DISCUSSION 29

V. CONCLUSION 33

REFERENCES 34

국문요약 43
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dc.description.tableofcontentsLIST OF FIGURES

Fig. 1. NT-3 overexpressing human neural stem cells 13

Fig. 2. Seeding human neural stem cells (hNSCs) in PLGA Scaffold 14

Fig. 3. Implantation of PLGA seeded with hNSCs (PLGA-NT3.F3) 16

Fig. 4. Area quantifications of spared tissue and residual scaffold 17

Fig. 5. Detection of transplanted human neural stem cells (hNSCs) 19

Fig. 6. Detection of transplanted human neural stem cells (hNSCs) in white matter 20

Fig. 7. Differentiation of neural stem cells in white matter 21

Fig. 8. Deposition of chondroitin sulfate proteoglycans (CSPGs) at the interface 23

Fig. 9. Detection of serotonergic neuron in lesion site by 5-HT staining 26

Fig. 10. Quantification of serotonergic neurons in caudal area : 5-HT staining 27
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dc.description.tableofcontentsLIST OF ABBREVIATION

APC-CC1, anti-APC (Ab-7) mouse mAb (CC-1)

BDNF, brain-derived neurotrophic factor

CNS, central nervous system

CS-56, anti-chondroitin sulfate

CSPG, chondroitin sulfate proteoglycans

DAPI, 4’, 6 –Diamidino-2-Phenylindole Dihydrochloride

DMEM, dulbecco’s modified eagle’s medium

DW, distilledwater

ELISA, enzyme-linked immunosorbent assay

EM, electron microscope

F3, human brain F3

GFAP, anti-glial fibrillary acidic protein

hNSC, human neural stem cell

5-HT, 5-hydroxytryptamine

Hu-Mito, anti-human mitochondria

L, left

NGF, nerve growth factor

NSAID, nonsteroidal antiinflammatory drug

NT3, neurotrophin-3

NT3.F3, neurotrophin-3 overexpressing F3

MAP2, anti-microtubule-associated protein 2

PBS, phosphate buffered saline

PLA, poly lactic acid

PLGA, poly (lactic-co-glycolic acid)

PGA, poly (glycolic acid)

P/S, penicillin streptomycin

R, right

ROI, regions of interest

RT, room temperature

S or SC, scaffold

SCI, spinal cord injury

T, tissue

VH, ventral horn

WM, white matter





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dc.language.isoen-
dc.titleDevelopment of Combinatorial Strategies Employing Neural Stem Cells and Polymer Scaffold for Spinal Cord Repair-
dc.title.alternative척수재생을 위한 신경줄기세포와 고분자 지지체를 이용한 복합적인 치료전략의 개발-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000009802-
dc.subject.keywordspinal cord injury-
dc.subject.keywordneural stem cell-
dc.subject.keywordneurotrophin-3-
dc.subject.keywordpoly (lactic-co-glycolic acid) scaffolds-
dc.subject.keywordserotonergic axon-
dc.description.degreeMaster-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor강, 영미-
dc.date.awarded2009-
dc.type.localTheses-
dc.citation.date2009-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
Appears in Collections:
Theses > Graduate School of Biomedical Sciences > Master
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