Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium

Abstract

The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal-type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation.

Niche regulation of corneal epithelial stem cells at the limbus

“…For example, limbal SCs have the smallest cell size [5], are slow-cycling and hence label-retaining [6], and do not express markers destined for terminal differentiation such as cytokeratins 3 [1] and 12 [7][8][9], involucrin [10], and connexin 43 [11]. In contrast, the SC-containing limbal epithelium has a high proliferative potential in different cultures [12][13][14][15], and their in vitro proliferation is resistant to the inhibition by tumor-promoting phorbol esters [13,16,17].…”

Intrastromal Invasion by Limbal Epithelial Cells Is Mediated by Epithelial-Mesenchymal Transition Activated by Air Exposure

“…2 The SCs of the corneal epithelium are located in the basal epithelial layer of the limbus between the cornea and the conjunctiva, whereas the TACs are located at the corneal basal epithelium. 3 Such a unique compartmentalization facilitates studies showing differences between limbal SC and corneal TAC in the expression of keratins 3 and 12, [3][4][5] connexin 43 (Cx43)-mediated gap junction intercellular communication, 6 and the nuclear transcription factor of p63, 7 the cell cycle length, 8 differential responses to tumorpromoting phorbol esters, 9 cell size, 10 and ex vivo expansion supported by 3T3 fibroblast feeder layers 11,12 or by amniotic membrane. 13 The homeostasis of the corneal epithelium is ultimately maintained by limbal epithelial SCs.…”

Keratolimbal allograft in corneal reconstruction

“…1,2 The existence of these limbal epithelial SC is supported by the following five well-established facts: (1) These cells do not express such corneal epithelial differentiation markers as keratin 3, 1 keratin 12, [3][4][5] and connexin 43. 6,7 (2) Epithelial neoplasias are commonly located in the limbal area.…”