Factors modulating p63 expression in cultured limbal epithelial cells

Hani Salehi-Had, Lenio S. Alvarenga, Roslyn Rivkah Isseroff, Aivan R. Schwab

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Purpose: The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. Methods: Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) × 104 cells/cm2 and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. Results: More than 85% (85%-90%) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4%), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9%). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. Conclusions: The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.

Original languageEnglish (US)
Pages (from-to)845-852
Number of pages8
JournalCornea
Volume24
Issue number7
DOIs
StatePublished - Oct 2005

Fingerprint

Epithelial Cells
Keratin-3
Starvation
Stem Cells
Cell Division
Cell Count
Differentiation Antigens
Intercellular Signaling Peptides and Proteins
Analysis of Variance
Transcription Factors

Keywords

  • Cell-cell contact
  • Corneal epithelial differentiation
  • Corneal epithelial stem cells
  • Cultured corneal limbal epithelial cells
  • p63

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Factors modulating p63 expression in cultured limbal epithelial cells. / Salehi-Had, Hani; Alvarenga, Lenio S.; Isseroff, Roslyn Rivkah; Schwab, Aivan R.

In: Cornea, Vol. 24, No. 7, 10.2005, p. 845-852.

Research output: Contribution to journalArticle

Salehi-Had, Hani ; Alvarenga, Lenio S. ; Isseroff, Roslyn Rivkah ; Schwab, Aivan R. / Factors modulating p63 expression in cultured limbal epithelial cells. In: Cornea. 2005 ; Vol. 24, No. 7. pp. 845-852.
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abstract = "Purpose: The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. Methods: Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) × 104 cells/cm2 and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. Results: More than 85{\%} (85{\%}-90{\%}) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4{\%}), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9{\%}). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. Conclusions: The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.",
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T1 - Factors modulating p63 expression in cultured limbal epithelial cells

AU - Salehi-Had, Hani

AU - Alvarenga, Lenio S.

AU - Isseroff, Roslyn Rivkah

AU - Schwab, Aivan R.

PY - 2005/10

Y1 - 2005/10

N2 - Purpose: The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. Methods: Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) × 104 cells/cm2 and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. Results: More than 85% (85%-90%) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4%), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9%). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. Conclusions: The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.

AB - Purpose: The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. Methods: Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) × 104 cells/cm2 and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. Results: More than 85% (85%-90%) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4%), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9%). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. Conclusions: The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.

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