cell sheet harvest
TRANSCRIPT
Kohji Nishida, M.D., Ph.D.
Department of Ophthalmology
Tohoku University Graduate
School of Medicine
Corneal Reconstruction Using Tissue-Engineered Epithelial Cell Sheets
Fabricated ex vivo From Autologous Oral Mucosal Epithelium
OsakaTokyo
Sendai
Stem Cells Scafold
from Vacanti & Langer Science 1993
Growth Factor
Concept of Regenerative Medicine Concept of Regenerative Medicine (Tissue Engineering)(Tissue Engineering)
Tissue Stem CellsTissue Stem Cells
3E. Fuchs, Cell, 2000
Epidermal stem cell Hair follicel stem cells
Gastrointestinal stem cells Neural stem cells Hematopooetic stem cells
brain
heartliver
kidney
bone
eye
spleen
muscle
pancreas
Pluripotent Stem CellsPluripotent Stem Cells
Embryonic stem cells induced pluripotent stem cells
epithelium
stroma
Endothelial disease
Alkali, thermal burnStvens-Johnson syndrome
Corneal dystrophy
Bullous keratopathy
Corneal epithelial disease
Stromal disease
endothelium
Standard therapy: allogeneic corneal transplanation using eye bank cornea
Corneal DiseasesCorneal Diseases
conjunctiva limbuscornea
cornea
conjunctiva
limbusBasal cells of Limbal epithelium
=stem cells
Corneal Epithelial Stem CellCorneal Epithelial Stem Cell
7
epi
stromaendo
cornea
Conj.limbus
Stem cell
Chemical burnThermal burn
Stevens-JohnsonOcular cicatricial
pemphigoid
Idiopathic
Congenital Secondary
Primary
(Nishida K. Cornea 2003)
Aniridia
Conjunctivalization
Limbal stem cell deficiencyLimbal stem cell deficiency
Corneal epithelial stem cellsCorneal epithelial stem cells
Normal corneaThermalalkali injury
SJS
Limbal stem cell deficiency
Long-Term Results of Corneal Transplantation for Corneal Epithelial Diseases
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(Solomon et al,
Ophthalmology, 2002)
Chem BurnSJSOther
0 20 40 60 80 100 1200
20
40
60
80
100
Months
Gra
ft S
urvi
val(
%)
High Risk of Rejection
SJSOCP SJS
Organ/tissue Transplant
Autologous epithelial cell sheet transplantation
Regenerative Medicine
Corneal transplant
RejectionLimited number of Donor cornea
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Cultured corneal epithelial sheet harvested by dispasetreatment
Initial ReportInitial Report
Pellegrini et al. Lancet 1997
Cell source: Corneal epithelial stem cell
The problems of Previous MethodsThe problems of Previous Methods
Cell sheet harvest by dispase
Cell source: corneal epithelial stem cellsApplied only to unilateral disease
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confluent cells
enzymaticproteolysis
marked degradation of cell-to-cell junction and ECM ⇒fragile cell sheet
confluent cells
enzymaticproteolysis
reducing temperature
Retain cell-cell junctions as well as deposited extracellular matrix below the lower cell membranes
Cell Sheet Harvest
confluent cells
enzymaticproteolysis
reducing temperature
Retain cell-cell junctions as well as deposited extracellular matrix below the lower cell membranes
Cell Sheet Harvest
Electron beamElectron beamTemperature-responsivemonomer
Temperature-responsivepolymer
• 37℃ hydrophobic ⇒ cell attachment
• below 32℃ hydrophilic ⇒ cell detachment
Cell sheet harvest IssueCell sheet harvest IssueUse of Temperature responsive culture dishUse of Temperature responsive culture dish
Cell attachment and detachment Cell attachment and detachment by temperature changeby temperature change
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ディスパーゼ処理
2mmX2mm
Limbal epithelium
Tissue-engineered epithelial cell sheet
Temperature-responsiveculture dish
37ºC culture2 weeks
20ºC
harvest
MMC-3T3 feeder
(Nishida K, Transplantation 2004)
NEW TECHNOLOGY: tissue-engineered epithelial cell sheet tranplantation
Cultivated corneal sheet
Cultivated oral mucosal sheet
Cell sheet harvest
K3
H&E
Characteristics of cultivated human corneal epithelial sheet
Normal cornea
apical
basal
(Nishida K, Transplantation 2004)
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Unilateral diseases Bilateral diseases
Autologous cell soruce Autologous cell soruce
(Nishida K et al. Transplantaion, 2004) (Nishida K, et al. N Engl J Med 2004)
Transplantation of tissueTransplantation of tissue--engineered epithelial engineered epithelial cell sheet for corneal diseasescell sheet for corneal diseases
Oral mucosal epithelial cell sheet
Normal cornea
Normal oral mucosa
Corneal epithelial cell sheet(Nishida K et al. Transplantaion, 2004)
(Nishida K, et al. N Engl J Med 2004)
Histology
Immunostaining for MUC 16Oral mucosal epithelium (in vivo)Corneal epithelium (in vivo)
Oral mucosal cell sheetCorneal epithelial cell sheet
(Hori Y, et al Exp Eye Res 2008)
Clinical ApplicationSubjects
Severe total limbal stem cell deficiency: Stevens-Johnson syndrome, Ocular cicatricialpemphigoid, Chemical burn, Thermal burn, Aniridia, etc
Surgery Cell sheet transplantation using autologous cell soruce
Post-operative managementTopical administration of 0.3 % levofuloxacin and 0.1 % betamethasone four times dailySystemic administration of betamethasone (1-2 mg/day) for the first two weeks after surgery
Pre-op 14 months post-op
VA=20/2000 VA=20/25
CASE Autologous Oral Epithelial Cell SheetTransplantation to Treat Ocular Pemphigoid
Results of Initial 4 CasesResults of Initial 4 Cases
(Nishida K, et al. N Engl J Med 2004)
CASE Autologous Oral Epithelial Cell Sheet Transplantation with PKP (epithelium scraped off) to Treat
Stevens-Johnson syndrome (post PKP)
VA=HM VA=20/20
Pre-op 3 year post-op
CASE Autologous Epithelial Cell SheetTransplantation with LKP (epithelium scraped off) and AMT to Treat Severe
Thermal burn
VA=HM VA=20/200
Pre-op 2 year post-op
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20/2000
20/2000 20/200 20/20LP
20/200
20/20
Surgical ResultsBest-Corrected Visual Acuity
CF
CF
HD
HD
Pre-op visual acuity
Post
-op
visu
al a
cuity
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3 years post-opepreope
Neovascularization
Noneovascularization
Corneal Neovascularization Oral mucosal epithelial cell sheet induce peripheral neovascularization in some cases
Issues to be solvedIssues to be solved
2 years post-ope
(N=4 *; P<0.05,Mann-Whitney test)
Quantification of Angiogenesis-Related Factors in Conditioned Medium from corneal sheet and oral sheet by ELISA
• FGF2 was significantly higher in oral sheet than in corneal sheet., indicating that FGF2 is a candidate involved in the induction of corneal neovascularization after oral sheet transplantation.
• Anti-FGF2 therapy may control the neovascularization after oral sheet transplantation.
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HE染色
Transplantationcontrol
Development of cultured corneal endothelial cell sheet transplantation
Epithelium
Stroma
endothelium
1.culture2. Fabrication and harvest of cultured
corneal endothelial cell sheet
3. Transplantation surgery Corneal thickness
(Ide T, Nishida K, et al Biomaterials 2005)(Sumide T, Nishida K, et al FASEB J 2006)
Cell density>3000/mm2
Autologous cells
上皮前駆細胞
角膜内皮細胞
iPS細胞
Corneal endothelial cells
Corneal diseases
iPS cells production
Corneal epithelial cells
培養細胞シート移植
角膜内皮シート
Autologoustranslation
角膜上皮
角膜内皮
難治性角膜疾患培養細胞シートの作製
角膜上皮シート
遺伝子導入遺伝子導入((Nanog, Sox2, Nanog, Sox2,
Klf4Klf4等)等)
culture
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1. Clinical application of cell sheet transplantation using autologous limbal or oral mucosal epithelial stem cells are effective for reconstructing the corneal surface.
2. Corneal neovasucularization induced in peripheral cornea after oral mucosal cell sheet grafting should be controlled.
Conclusions
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AcknowledgementAcknowledgement
Department of Ophthalmology, Osaka University Medical School
Soma T, Hayashida Y, WatanabeK, Ide T, Sumide T, Saito T, Kaneyama S, Maeda N, Watanabe H, Hori Y, Tano Y
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical UniversityYamato M, Kikuchi A, Yang J,
Okano T
Department of Ophthalmology, Tohoku UniversityKubota A, Hayashi R, Takayanagi T, Yokokura R, Kanakubo A, Kikuchi M
Ryu T
Osaka
Tokyo