Human limbal progenitor cell characteristics are maintained in tissue culture

Shaohui Liu, Jing Li, Chuanfu Wang, Donald Tan, Roger Beuerman

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Introduction: To determine the differentiation of human limbal epithelial cells in tissue culture. Materials and Methods: Epithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR). Results: The colony-forming efficiency of primary cultured (P0) cells was about 19.35 ± 6.46% (mean ± SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P >< 0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 ± 1.27)% (n = 7, P0) and (3.96 ± 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse. Conclusions: Human limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.

Original languageEnglish (US)
Pages (from-to)80-86
Number of pages7
JournalAnnals of the Academy of Medicine Singapore
Volume35
Issue number2
StatePublished - Feb 1 2006
Externally publishedYes

Fingerprint

Stem Cells
Keratin-12
Nuclear Antigens
Connexin 43
Epithelial Cells
Real-Time Polymerase Chain Reaction
Cultured Cells
Keratin-3
Cell Proliferation
Staining and Labeling
Feeder Cells
Mitomycin
Cornea
Coloring Agents
Immunohistochemistry
Air
Phenotype
Messenger RNA
involucrin

Keywords

  • Cell culture
  • Feeder layer
  • Limbal epithelial cells
  • Progenitor cells
  • Stem cells

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Human limbal progenitor cell characteristics are maintained in tissue culture. / Liu, Shaohui; Li, Jing; Wang, Chuanfu; Tan, Donald; Beuerman, Roger.

In: Annals of the Academy of Medicine Singapore, Vol. 35, No. 2, 01.02.2006, p. 80-86.

Research output: Contribution to journalArticle

Liu, Shaohui ; Li, Jing ; Wang, Chuanfu ; Tan, Donald ; Beuerman, Roger. / Human limbal progenitor cell characteristics are maintained in tissue culture. In: Annals of the Academy of Medicine Singapore. 2006 ; Vol. 35, No. 2. pp. 80-86.
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abstract = "Introduction: To determine the differentiation of human limbal epithelial cells in tissue culture. Materials and Methods: Epithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR). Results: The colony-forming efficiency of primary cultured (P0) cells was about 19.35 ± 6.46{\%} (mean ± SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P >< 0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 ± 1.27){\%} (n = 7, P0) and (3.96 ± 1.35){\%} (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse. Conclusions: Human limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.",
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AU - Liu, Shaohui

AU - Li, Jing

AU - Wang, Chuanfu

AU - Tan, Donald

AU - Beuerman, Roger

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N2 - Introduction: To determine the differentiation of human limbal epithelial cells in tissue culture. Materials and Methods: Epithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR). Results: The colony-forming efficiency of primary cultured (P0) cells was about 19.35 ± 6.46% (mean ± SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P >< 0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 ± 1.27)% (n = 7, P0) and (3.96 ± 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse. Conclusions: Human limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.

AB - Introduction: To determine the differentiation of human limbal epithelial cells in tissue culture. Materials and Methods: Epithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR). Results: The colony-forming efficiency of primary cultured (P0) cells was about 19.35 ± 6.46% (mean ± SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P >< 0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 ± 1.27)% (n = 7, P0) and (3.96 ± 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse. Conclusions: Human limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.

KW - Cell culture

KW - Feeder layer

KW - Limbal epithelial cells

KW - Progenitor cells

KW - Stem cells

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