April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Regeneration of corneal epithelium with conjunctival epithelial equivalents generated in serum and feeder cell-free medium
Author Affiliations & Notes
  • So-Hyang Chung
    Ophthalmology, The Catholic University of Korea, Seoul, Republic of Korea
  • Sohee Jeon
    Medical Science, The Catholic University of Korea, Seoul, Republic of Korea
    Catholic High Performance Cell Therapy Center, The Catholic University of Korea, Seoul, Republic of Korea
  • Seong Hyun Choi
    Ophthalmology, The Catholic University of Korea, Seoul, Republic of Korea
  • Jose Wolosin
    Icahn School of Medicine at Mount Sinai, New York, NY
  • Choun-Ki Joo
    Ophthalmology, The Catholic University of Korea, Seoul, Republic of Korea
  • Footnotes
    Commercial Relationships So-Hyang Chung, None; Sohee Jeon, None; Seong Hyun Choi, None; Jose Wolosin, None; Choun-Ki Joo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5160. doi:
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      So-Hyang Chung, Sohee Jeon, Seong Hyun Choi, Jose Wolosin, Choun-Ki Joo; Regeneration of corneal epithelium with conjunctival epithelial equivalents generated in serum and feeder cell-free medium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5160.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it will be desirable to eliminate the involvement of xenogeneic components, such as non-human sera and feeder cell. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3- free culture conditions when transplanted into the de-epithelialized limbal corneal surface.

 
Methods
 

Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low calcium medium, and set in air-liquid interface culture for 14 days. Resulting multi-layered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry.

 
Results
 

At graft time the cultured epithelial sheet consisted of 6-8 layers of properly stratified epithelium that displayed a CK19+/MUC5AC+/ CK3 -/ CK12- phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing an established anti-human nuclei antibody, the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival Goblet cell mucin (MUC5AC) was not visible.

 
Conclusions
 

Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3- free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.

 
 
Immunohistochemical examinations of the cultivated human conjunctival epithelial (CjE) equivalent. The cultivated human CjE equivalent were stained green for MUC5AC (A), CK19 (B), CK3 (C), CK12 (D), red for p63 (E) by indirect immunofluorescence. Nuclei has been counterstained with DAPI. Arrow points to positive cell for MUC5AC (A).
 
Immunohistochemical examinations of the cultivated human conjunctival epithelial (CjE) equivalent. The cultivated human CjE equivalent were stained green for MUC5AC (A), CK19 (B), CK3 (C), CK12 (D), red for p63 (E) by indirect immunofluorescence. Nuclei has been counterstained with DAPI. Arrow points to positive cell for MUC5AC (A).
 
 
Immunohistochemistry of the engrafted human conjunctival epithelial equivalent. A. Stain with anti-human nuclear antibody B. p63 with DAPI counterstain. C. Ki67-DAPI. D. Integrin β4-DAPI. E. MUC1-DAPI. F. MUC5AC-DAPI. G. CK3-DAPI. H. CK12-DAPI.
 
Immunohistochemistry of the engrafted human conjunctival epithelial equivalent. A. Stain with anti-human nuclear antibody B. p63 with DAPI counterstain. C. Ki67-DAPI. D. Integrin β4-DAPI. E. MUC1-DAPI. F. MUC5AC-DAPI. G. CK3-DAPI. H. CK12-DAPI.
 
Keywords: 687 regeneration • 482 cornea: epithelium • 474 conjunctiva  
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