Limits...
Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction.

Madhira SL, Vemuganti G, Bhaduri A, Gaddipati S, Sangwan VS, Ghanekar Y - Mol. Vis. (2008)

Bottom Line: Ultrastructural studies were also performed using electron microscopy.Electron microscopy demonstrated that the cells formed gap junctions and desmosomes.The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

View Article: PubMed Central - PubMed

Affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory,L.V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction.

Methods: Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy.

Results: A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

Conclusions: Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.

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Ultrastructural studies of limbal, oral, and conjunctival cultures. A: The limbal epithelial cell is shown with the desmosomes (D) and tight junction (TJ) revealed in the inset. B: Oral epithelial cell is also shown with the desmosomes (D) and gap junction (GJ) revealed in the inset. C: Conjunctival epithelial cell is shown with just the desmosomes (D) demonstrated in the inset.
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f2: Ultrastructural studies of limbal, oral, and conjunctival cultures. A: The limbal epithelial cell is shown with the desmosomes (D) and tight junction (TJ) revealed in the inset. B: Oral epithelial cell is also shown with the desmosomes (D) and gap junction (GJ) revealed in the inset. C: Conjunctival epithelial cell is shown with just the desmosomes (D) demonstrated in the inset.

Mentions: Electron microscopy was used to check the presence of cell-cell junctions and cell-basement membrane junctions. As shown in Figure 2B, cultured oral epithelial cells were able to form gap junctions and desmosomes with each other, which is similar to limbal and conjunctival cultures. However, hemidesmosomes, the junctions between cells and the amniotic membrane, were not clearly visible in any of these cultures under this resolution, although cells from all three cultures were in close apposition with the amniotic membrane (data not shown).


Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction.

Madhira SL, Vemuganti G, Bhaduri A, Gaddipati S, Sangwan VS, Ghanekar Y - Mol. Vis. (2008)

Ultrastructural studies of limbal, oral, and conjunctival cultures. A: The limbal epithelial cell is shown with the desmosomes (D) and tight junction (TJ) revealed in the inset. B: Oral epithelial cell is also shown with the desmosomes (D) and gap junction (GJ) revealed in the inset. C: Conjunctival epithelial cell is shown with just the desmosomes (D) demonstrated in the inset.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254962&req=5

f2: Ultrastructural studies of limbal, oral, and conjunctival cultures. A: The limbal epithelial cell is shown with the desmosomes (D) and tight junction (TJ) revealed in the inset. B: Oral epithelial cell is also shown with the desmosomes (D) and gap junction (GJ) revealed in the inset. C: Conjunctival epithelial cell is shown with just the desmosomes (D) demonstrated in the inset.
Mentions: Electron microscopy was used to check the presence of cell-cell junctions and cell-basement membrane junctions. As shown in Figure 2B, cultured oral epithelial cells were able to form gap junctions and desmosomes with each other, which is similar to limbal and conjunctival cultures. However, hemidesmosomes, the junctions between cells and the amniotic membrane, were not clearly visible in any of these cultures under this resolution, although cells from all three cultures were in close apposition with the amniotic membrane (data not shown).

Bottom Line: Ultrastructural studies were also performed using electron microscopy.Electron microscopy demonstrated that the cells formed gap junctions and desmosomes.The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

View Article: PubMed Central - PubMed

Affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory,L.V. Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction.

Methods: Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy.

Results: A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.

Conclusions: Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.

Show MeSH
Related in: MedlinePlus