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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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Expression of markers of epithelial stem cells and differentiation. RT-PCR analysis of expression of markers for epithelial differentiation (A) and stem cells (B) is shown. GAPDH was used as the normalizing control. The RT-PCR results of cytokeratin K5 and connexin 43 show a composite picture of PCR of cDNA from all three cells and the negative control that were performed in the same experiment. All experiments were performed twice with RNA isolated from a separate set of cultures.
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f3: Expression of markers of epithelial stem cells and differentiation. RT-PCR analysis of expression of markers for epithelial differentiation (A) and stem cells (B) is shown. GAPDH was used as the normalizing control. The RT-PCR results of cytokeratin K5 and connexin 43 show a composite picture of PCR of cDNA from all three cells and the negative control that were performed in the same experiment. All experiments were performed twice with RNA isolated from a separate set of cultures.

Mentions: Expression of different markers for stem cells as well as differentiated epithelial cells in cultured oral, limbal, and conjunctival cells was checked by reverse-transcription polymerase chain reaction (RT–PCR) analysis. cDNA was synthesized from RNA isolated from confluent cultures and was subjected to PCR using the primers shown in Table 1. Both limbal and oral epithelial cells expressed cytokeratin K3, the marker for differentiated corneal epithelial cells (Figure 3A). The oral epithelial cultures did not express cytokeratin K12, indicating that the cells retained the phenotype of oral epithelial cells. Interestingly, conjunctival cultures also expressed cytokeratin K3 and K12. The cells also expressed cytokeratin K4 and K13, markers of nonkeratinized stratified oral epithelia [23,24] as seen earlier in rabbit oral epithelial cultures [16]. These cytokeratins were also expressed by cultured limbal and conjunctival cultures. Cytokeratin K15, expressed in basal and suprabasal cells of the limbus [25], was also observed in all three cultured cells. Connexin 43, another marker for differentiated epithelial cells, was also expressed by these cells. Cultured oral cells did not express Pax-6, a marker for ocular tissues.


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)

Expression of markers of epithelial stem cells and differentiation. RT-PCR analysis of expression of markers for epithelial differentiation (A) and stem cells (B) is shown. GAPDH was used as the normalizing control. The RT-PCR results of cytokeratin K5 and connexin 43 show a composite picture of PCR of cDNA from all three cells and the negative control that were performed in the same experiment. All experiments were performed twice with RNA isolated from a separate set of cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Expression of markers of epithelial stem cells and differentiation. RT-PCR analysis of expression of markers for epithelial differentiation (A) and stem cells (B) is shown. GAPDH was used as the normalizing control. The RT-PCR results of cytokeratin K5 and connexin 43 show a composite picture of PCR of cDNA from all three cells and the negative control that were performed in the same experiment. All experiments were performed twice with RNA isolated from a separate set of cultures.
Mentions: Expression of different markers for stem cells as well as differentiated epithelial cells in cultured oral, limbal, and conjunctival cells was checked by reverse-transcription polymerase chain reaction (RT–PCR) analysis. cDNA was synthesized from RNA isolated from confluent cultures and was subjected to PCR using the primers shown in Table 1. Both limbal and oral epithelial cells expressed cytokeratin K3, the marker for differentiated corneal epithelial cells (Figure 3A). The oral epithelial cultures did not express cytokeratin K12, indicating that the cells retained the phenotype of oral epithelial cells. Interestingly, conjunctival cultures also expressed cytokeratin K3 and K12. The cells also expressed cytokeratin K4 and K13, markers of nonkeratinized stratified oral epithelia [23,24] as seen earlier in rabbit oral epithelial cultures [16]. These cytokeratins were also expressed by cultured limbal and conjunctival cultures. Cytokeratin K15, expressed in basal and suprabasal cells of the limbus [25], was also observed in all three cultured cells. Connexin 43, another marker for differentiated epithelial cells, was also expressed by these cells. Cultured oral cells did not express Pax-6, a marker for ocular tissues.

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