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

8

(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

10

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

11

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

13

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)

17

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

27

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

28

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.

30

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

32

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

33

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