the oral mucosal biopsy , in vitro culture of oral mucosal epithelium and the surgical transplantations were done as reported previously . this study adhered to the tenets of the declaration of helsinki and was approved by the institutional review board . on the 1 day and after 11 months of comet for the right eye [ fig . on the 1 post - operative day of pk , the uncorrected visual acuity was 20/60 improving to 20/40 . when last seen at 13 months post - pk , the best corrected visual acuity was 20/160 with a clear graft and a stable ocular surface . slit lamp photograph of the right eye showing total vascularized cornea due to limbal stem cell deficiency ( a ) slit lamp photograph , post - operative day 1 ( b ) and after 10 months ( c ) post- comet and post comet - pk ( d ) slit lamp pictures h and e staining of the corneal button , excised during pk of the comet - treated right eye , showed a 5 to 6 cell stratified epithelium with basement membrane . goblet cells were not observed in pas staining , thereby suggesting the absence of conjunctivalization . consistent with the previous reports , a few sub - epithelial vasculatures were also seen in close proximity to the basement membrane [ figs . 2c and d ] . growth pattern of cultivated oral mucosal epithelial cells on denuded ham and histological pictures of post - comet cornea . ( a ) phase contrast images taken at day 2 in culture showing the initiation of cellular outgrowth from the explant tissue and monolayer formation ( arrows ) . ( c ) hematoxylin and eosin , pas staining on post comet pk tissue showing hyperplasia of epithelial cells . ( d ) pas staining shows no goblet cells in the stratified epithelium both corneal and oral mucosal tissues share the expression of some of the cytokeratins like k3 , k4 , k13 and k15 with the conjunctival epithelium . immunohistochemical ( ihc ) examination of the post - comet corneal tissue showed k19 being expressed in all the layers of conjunctival epithelium while it was expressed only by the basal cells of the cornea , oral mucosal tissue and in the post - comet corneal button [ figs . expression of k14 was seen only in the basal cells of the conjunctiva but not in the cornea , oral mucosa and in the post - comet corneal tissues [ figs . d ] . while the epithelial cells of all the layers of the native cornea and the pk button stained positive for the antibody that collectively recognizes the cytokeratins , k3/k12 [ fig . l ] , the k12-specific antibody stained only the basal cells of the pk button with a clear cytosolic expression pattern [ fig . 3m k14 was not expressed by the epithelial cells of the ( a ) central cornea , ( b ) oral mucosal epithelium and ( d ) post - comet corneal tissue , but were expressed by the ( c ) basal conjunctival epithelial cells ( arrow head ) . k19 is expressed in all the layers of the limbal ( l ) ( arrow ) ( e ) and conjunctival epithelium ( g ) but not expressed in the central cornea ( data not shown ) . the basal cells of oral mucosal epithelium ( arrow ) ( f ) and the post comet pk tissue showed k19 expression . k 3/12 staining is present in central corneal epithelium ( i ) , oral mucosal epithelium ( j ) and post - comet corneal epithelium ( l ) but not present in conjunctiva ( k ) . pax6 was clearly expressed in nucleus of the central corneal epithelium ( m ) and conjunctival epithelium ( o ) . the post comet pk tissue ( p ) showed a clear increased pax6 nuclear staining in the basal ( arrow ) and supra - basal cells . k12 is expressed throughout the central corneal epithelium ( q ) and is expressed faintly in the cytoplasm of the basal cells ( arrow in 3 t ) of the post - comet corneal tissue ( t ) , oral mucosal tissue ( r ) and conjunctival epithelium do not show any cytoplasm staining . consistent nuclear reactivity ( may be non - specific ) was also noted with k12 antibody only in the oral and comet tissues ( all pictures magnification- 400 , 3 t and 3r-1000 ) to our surprise , we found pax6 expression in the ihc sections of the native oral tissue ( diffused and pan - nuclear pattern ) as opposed to the strong nuclear staining in the corneal and conjunctival epithelium [ fig . more interestingly , the basal cells of the post - comet corneal button showed a clear increase in the nuclear pax6 staining [ fig . , we found that the supra - basal cells were positive for the cell proliferation marker ki-67 , similar to the native oral , corneal , and conjunctival tissues [ fig . the basal and suprabasal cells of post - comet corneal tissue showed reactivity to the epithelial stem cell marker , p63 [ fig . l ] , thus confirming the presence of stem cells and ta cells in the reconstructed ocular surface . 2c ] endothelial cells express cd31 and cd34 [ fig . 5 and b ] . staining with ki-67 antibody was also positive in endothelial cells [ fig . immunohistochemistry for ki 67 in corneal epithelium ( a ) , oral mucosal epithelium ( b ) , conjunctival epithelium ( c ) , post comet pk corneal ( d ) tissue showed clear nuclear expression by the proliferating supra - basal cells . in the post comet pk corneal tissue , ki67 expression was also noted in the nucleus of some of the endothelial cells lining the sub epithelial vasculatures ( arrow in d ) . p63 immunostaining showed nuclear staining in the basal and supra - basal cells of all the tissues tested ( e - h ) . p75 immunostaining showed membrane staining only in the basal epithelial cells of the cornea ( i ) , oral mucosa ( j ) , conjunctiva ( k ) and post comet pk tissue ( l ) . ( magnification for ki 67 and p63- 400 , p75- 1000 ) endothelial markers for blood capillaries . immunohistochemistry for cd31 ( a ) , cd34 ( b ) of post comet pk tissue showed positivity in all subepithelial vasculature ( stars ) ( magnification -400 ) on the 1 day and after 11 months of comet for the right eye [ fig . on the 1 post - operative day of pk , the uncorrected visual acuity was 20/60 improving to 20/40 . when last seen at 13 months post - pk , the best corrected visual acuity was 20/160 with a clear graft and a stable ocular surface . slit lamp photograph of the right eye showing total vascularized cornea due to limbal stem cell deficiency ( a ) slit lamp photograph , post - operative day 1 ( b ) and after 10 months ( c ) post- comet and post comet - pk ( d ) slit lamp pictures h and e staining of the corneal button , excised during pk of the comet - treated right eye , showed a 5 to 6 cell stratified epithelium with basement membrane . goblet cells were not observed in pas staining , thereby suggesting the absence of conjunctivalization . consistent with the previous reports , a few sub - epithelial vasculatures were also seen in close proximity to the basement membrane [ figs . 2c and d ] . growth pattern of cultivated oral mucosal epithelial cells on denuded ham and histological pictures of post - comet cornea . ( a ) phase contrast images taken at day 2 in culture showing the initiation of cellular outgrowth from the explant tissue and monolayer formation ( arrows ) . ( c ) hematoxylin and eosin , pas staining on post comet pk tissue showing hyperplasia of epithelial cells . both corneal and oral mucosal tissues share the expression of some of the cytokeratins like k3 , k4 , k13 and k15 with the conjunctival epithelium . immunohistochemical ( ihc ) examination of the post - comet corneal tissue showed k19 being expressed in all the layers of conjunctival epithelium while it was expressed only by the basal cells of the cornea , oral mucosal tissue and in the post - comet corneal button [ figs . expression of k14 was seen only in the basal cells of the conjunctiva but not in the cornea , oral mucosa and in the post - comet corneal tissues [ figs . d ] . while the epithelial cells of all the layers of the native cornea and the pk button stained positive for the antibody that collectively recognizes the cytokeratins , k3/k12 [ fig . l ] , the k12-specific antibody stained only the basal cells of the pk button with a clear cytosolic expression pattern [ fig . 3m k14 was not expressed by the epithelial cells of the ( a ) central cornea , ( b ) oral mucosal epithelium and ( d ) post - comet corneal tissue , but were expressed by the ( c ) basal conjunctival epithelial cells ( arrow head ) . k19 is expressed in all the layers of the limbal ( l ) ( arrow ) ( e ) and conjunctival epithelium ( g ) but not expressed in the central cornea ( data not shown ) . the basal cells of oral mucosal epithelium ( arrow ) ( f ) and the post comet pk tissue showed k19 expression . k 3/12 staining is present in central corneal epithelium ( i ) , oral mucosal epithelium ( j ) and post - comet corneal epithelium ( l ) but not present in conjunctiva ( k ) . pax6 was clearly expressed in nucleus of the central corneal epithelium ( m ) and conjunctival epithelium ( o ) . the post comet pk tissue ( p ) showed a clear increased pax6 nuclear staining in the basal ( arrow ) and supra - basal cells . k12 is expressed throughout the central corneal epithelium ( q ) and is expressed faintly in the cytoplasm of the basal cells ( arrow in 3 t ) of the post - comet corneal tissue ( t ) , oral mucosal tissue ( r ) and conjunctival epithelium do not show any cytoplasm staining . consistent nuclear reactivity ( may be non - specific ) was also noted with k12 antibody only in the oral and comet tissues ( all pictures magnification- 400 , 3 t and 3r-1000 ) to our surprise , we found pax6 expression in the ihc sections of the native oral tissue ( diffused and pan - nuclear pattern ) as opposed to the strong nuclear staining in the corneal and conjunctival epithelium [ fig . more interestingly , the basal cells of the post - comet corneal button showed a clear increase in the nuclear pax6 staining [ fig . , we found that the supra - basal cells were positive for the cell proliferation marker ki-67 , similar to the native oral , corneal , and conjunctival tissues [ fig . the basal and suprabasal cells of post - comet corneal tissue showed reactivity to the epithelial stem cell marker , p63 [ fig . l ] , thus confirming the presence of stem cells and ta cells in the reconstructed ocular surface . 2c ] endothelial cells express cd31 and cd34 [ fig . 5 and b ] . staining with ki-67 antibody was also positive in endothelial cells [ fig . immunohistochemistry for ki 67 in corneal epithelium ( a ) , oral mucosal epithelium ( b ) , conjunctival epithelium ( c ) , post comet pk corneal ( d ) tissue showed clear nuclear expression by the proliferating supra - basal cells . in the post comet pk corneal tissue , ki67 expression was also noted in the nucleus of some of the endothelial cells lining the sub epithelial vasculatures ( arrow in d ) . p63 immunostaining showed nuclear staining in the basal and supra - basal cells of all the tissues tested ( e - h ) . p75 immunostaining showed membrane staining only in the basal epithelial cells of the cornea ( i ) , oral mucosa ( j ) , conjunctiva ( k ) and post comet pk tissue ( l ) . ( magnification for ki 67 and p63- 400 , p75- 1000 ) endothelial markers for blood capillaries . immunohistochemistry for cd31 ( a ) , cd34 ( b ) of post comet pk tissue showed positivity in all subepithelial vasculature ( stars ) ( magnification -400 ) this study reports a case of alkali burn - induced bilateral total lscd treated with autologous comet . in our study , the histological examination of the corneal button showed that the transplanted oral cells had undergone stratification and successfully reconstructed the ocular surface . it was encouraging to note that there was no recurrence of conjunctivalization as confirmed by pas staining , k14 and k19 expression patterns . the suprabasal cells were positive for the cell proliferation marker , ki-67 , thus confirming the presence of proliferating cells . interestingly , we found that the basal cells of the post - comet corneal button showed a clear increase in nuclear pax6 and cytoplasmic k12 expression . a recent report have shown full thickness k12 expression only in a peripheral zone of a comet - treated patient . wherein , the increased nuclear pax6 and the cytoplasmic k12 expression in the basal cells of the entire post - comet , pk button of our total lscd case is very encouraging . this suggests that the basal oral cells are responding to the signals from local corneal niche in vivo and are slowly acquiring the corneal phenotype . most of the basal and supra basal cells of the post - comet corneal tissue stained positive for p63 antibody against all 3 isoforms of p63 . though it confirms that the stem cells do survive and maintain the reconstructed tissue , they continue to reside in the basal layers of the post - comet corneal tissue , similar to the native oral tissue . in keeping with earlier reports , we observed subepithelial vasculatures in the corneal button that were positive for the endothelial markers , cd31 and cd34 . the post - operative inflammation and the vasculatures that persist post - debridement could have triggered the initial neovascularization until the ocular surface is stably reconstructed by the oral cells . the presence of blood cells in the vasculatures and ki-67 positive endothelial cells suggest that they are active vasculatures [ fig . a recent study reported trans - differentiation of hair follicle stem cells into corneal epithelial - like cells in limbal microenvironment . therefore , it is possible that these subepithelial vasculatures could slowly regress over time as the oral cells respond to the native corneal cues . we conclude that post - comet visual outcome depends on the severity of damage and the extent of stromal scarring , and can be further improved by an optical pk . the transplanted oral mucosal epithelial cells do survive and proliferate on the wounded corneal surface . while they maintain their native morphology and marker expression profiles at the ectopic site , they tend to respond to corneal cues in vivo and upregulate cornea - specific pax6 and k12 expression in the basal epithelial cells .
to understand the response of oral epithelial cells , transplanted on corneal surface to the ocular cues in vivo . the corneal button obtained after penetrating keratoplasty ( pk ) of an eye of a patient with total limbal stem cell deficiency ( lscd ) , previously treated with cultured oral mucosal epithelial transplantation ( comet ) 
 was examined by immunohistochemistry for the expression of keratins , p63 , p75 , pax6 , ki-67 , cd31 , and cd34 . 
 comet followed by optical - pk has improved visual acuity to 20/40 and rendered a stable ocular surface . 
 the excised corneal tissue showed the presence of stratified epithelium with vasculatures . 
 the epithelial cells of the corneal button expressed k3 , k19 , ki-67 , p63 , p75 and the cornea - specific pax6 and k12 . 
 this study confirms that the oral cells , transplanted to corneal surface , survive and stably reconstruct the ocular surface . 
 they maintain their stemness at the ectopic site and acquire some of the corneal epithelial - like characters .