Corneal Reconstruction Using Tissue- Engineered Epithelial Cell [PDF]

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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

Osaka Tokyo

Sendai

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Stem Cells Scafold

from Vacanti & Langer Science 1993

Growth Factor

Concept of Regenerative Medicine Concept of Regenerative Medicine (Tissue Engineering) (Tissue Engineering)

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Tissue Stem Cells Tissue Stem Cells

3 E . F u c h s , C e l l , 2

Epidermal stem cell Hair follicel stem cells Gastrointestinal stem cells Neural stem cells Hematopooetic stem cells

brain heart liver kidney bone eye spleen muscle pancreas

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Pluripotent Stem Cells Pluripotent Stem Cells

Embryonic stem cells induced pluripotent stem cells

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epithelium stroma

Endothelial disease

Alkali, thermal burn Stvens-Johnson syndrome Corneal dystrophy Bullous keratopathy

Corneal epithelial disease Stromal disease

endothelium

Standard therapy: allogeneic corneal transplanation using eye bank cornea

Corneal Diseases Corneal Diseases

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conjunctiva limbus cornea

cornea conjunctiv a limbus

Basal cells of Limbal epithelium =stem cells

Corneal Epithelial Stem Cell Corneal Epithelial Stem Cell

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epi stroma endo cornea Conj. limbus Stem cell

Chemical burn Thermal burn Stevens-Johnson Ocular cicatricial pemphigoid

Idiopathic Congenital Secondary Primary （Nishida K. Cornea 2003）

Aniridia Conjunctivalization

Limbal stem cell deficiency Limbal stem cell deficiency

Corneal epithelial stem cells Corneal epithelial stem cells

Normal cornea Thermal alkali injury SJS Limbal stem cell deficiency

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Long-Term Results of Corneal Transplantation for Corneal Epithelial Diseases

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(Solomon et al, Ophthalmology, 2002)

Chem Burn SJS Other

20 40 60 80 100 120 20 40 60 80 100

Months Graft Survival(% )

High Risk of Rejection

SJS OCP SJS

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Organ/ tissue Transplant

Autologous epithelial cell sheet transplantation

Regenerative Medicine

Corneal transplant

Rejection Limited number

f Donor cornea

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Cultured corneal epithelial sheet harvested by dispase treatment

Initial Report Initial Report

Pellegrini et al. Lancet 1997 Cell source: Corneal epithelial stem cell

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The problems of Previous Methods The problems of Previous Methods

Cell sheet harvest by dispase Cell source: corneal epithelial stem cells Applied only to unilateral disease

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confluent cells enzymatic proteolysis

marked degradation of cell-to-cell junction and ECM ⇒fragile cell sheet

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confluent cells enzymatic proteolysis reducing temperature

Retain cell-cell junctions as well as deposited extracellular matrix below the lower cell membranes

Cell Sheet Harvest

confluent cells enzymatic proteolysis reducing temperature

Retain cell-cell junctions as well as deposited extracellular matrix below the lower cell membranes

Cell Sheet Harvest

Electron beam Electron beam

Temperature-responsive monomer Temperature-responsive polymer

37℃ hydrophobic

⇒ cell attachment

below 32℃ hydrophilic

⇒ cell detachment

Cell sheet harvest Issue Cell sheet harvest Issue Use of Temperature responsive culture dish Use of Temperature responsive culture dish

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Cell attachment and detachment Cell attachment and detachment by temperature change by temperature change

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ディスパーゼ処理

2mmX2mm

Limbal epithelium Tissue-engineered epithelial cell sheet

Temperature-responsive culture dish

37º C culture 2 weeks 20º C

harvest

MMC-3T3 feeder (Nishida K, Transplantation 2004)

NEW TECHNOLOGY: tissue-engineered epithelial cell sheet tranplantation

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Cultivated corneal sheet Cultivated oral mucosal sheet

Cell sheet harvest

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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

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（ Nishida K et al. Transplantaion, 2004）（ Nishida K, et al. N Engl J Med 2004）

Transplantation of tissue Transplantation of tissue-

engineered epithelial

engineered epithelial cell sheet for corneal diseases cell sheet for corneal diseases

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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

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Immunostaining for MUC 16

Oral mucosal epithelium (in vivo)

Corneal epithelium (in vivo)

Oral mucosal cell sheet

Corneal epithelial cell sheet (Hori Y, et al Exp Eye Res 2008)

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Clinical Application

Subjects

Severe total limbal stem cell deficiency: Stevens-Johnson syndrome, Ocular cicatricial pemphigoid, Chemical burn, Thermal burn, Aniridia, etc

Surgery

Cell sheet transplantation using autologous cell soruce

Post-operative management

Topical administration of 0.3 % levofuloxacin and 0.1 % betamethasone four times daily Systemic administration of betamethasone (1-2 mg/day) for the first two weeks after surgery

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Pre-op 14 months post-op VA= 20/ 2000 VA= 20/ 25 CASE Autologous Oral Epithelial Cell Sheet Transplantation to Treat Ocular Pemphigoid

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Results of I nitial 4 Cases Results of I nitial 4 Cases

（Nishida K, et al. N Engl J Med 2004）

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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

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CASE Autologous Epithelial Cell Sheet Transplantation 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 20/20

LP

20/200 20/20

Surgical Results

Best-Corrected Visual Acuity

CF CF HD HD

Pre-op visual acuity Post-op visual acuity

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28 3 y e a r s p

s

t

p

e p r e

p

e Neovascularization No neovascularization

Corneal Neovascularization Oral mucosal epithelial cell sheet induce peripheral neovascularization in some cases

Issues to be solved Issues to be solved

2 y e a r s p

s

t

p

e

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(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

Transplantation control

Development of cultured corneal endothelial cell sheet transplantation

Epithelium Stroma endothelium

1.culture

2. Fabrication and harvest of cultured

corneal endothelial cell sheet

3. Transplantation surgery

Corneal thickness

(I de T, Nishida K, et al Biomaterials 2005) (Sumide T, Nishida K, et al FASEB J 2006)

Cell density > 3000/ mm2

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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

Autologous translat ion

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, Klf4 Klf4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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Acknowledgement Acknowledgement

Department of Ophthalmology, Osaka University Medical School Soma T, Hayashida Y, Watanabe K, I de T, Sumide T, Saito T, Kaneyama S, Maeda N, Watanabe H, Hori Y, Tano Y I nstitute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University Yamato M, Kikuchi A, Yang J, Okano T Department of Ophthalmology, Tohoku University Kubota A, Hayashi R, Takayanagi T, Yokokura R, Kanakubo A, Kikuchi M Ryu T

Osaka Tokyo