The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets

Masahiro Omoto, Hideyuki Miyashita, Shigeto Shimmura, Kazunari Higa, Tetsuya Kawakita, Satoru Yoshida, Michael Mcgrogan, Jun Shimazaki, Kazuo Tsubota

Research output: Contribution to journalArticle

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Abstract

PURPOSE. To report the efficacy of human bone marrow-de- rived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. METHODS. Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in -modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithe- lial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air- liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cyto- keratin 3 (K3), K15, p63, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and trans- planted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochem- istry against K3 and K4. RESULTS. MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. CONCLUSIONS. MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

Original languageEnglish
Pages (from-to)2109-2115
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number5
DOIs
Publication statusPublished - 2009

Fingerprint

Feeder Cells
Mesenchymal Stromal Cells
Keratin-3
Epithelial Cells
3T3 Cells
Rabbits
Bone Marrow
Immunohistochemistry
Fibroblast Growth Factor 7
Cell Engineering
Eagles
Hepatocyte Growth Factor
Mitomycin
Cadherins
Reverse Transcription
Histology
Stem Cells
Air
Cytokines
Transplants

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets. / Omoto, Masahiro; Miyashita, Hideyuki; Shimmura, Shigeto; Higa, Kazunari; Kawakita, Tetsuya; Yoshida, Satoru; Mcgrogan, Michael; Shimazaki, Jun; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 50, No. 5, 2009, p. 2109-2115.

Research output: Contribution to journalArticle

Omoto, Masahiro ; Miyashita, Hideyuki ; Shimmura, Shigeto ; Higa, Kazunari ; Kawakita, Tetsuya ; Yoshida, Satoru ; Mcgrogan, Michael ; Shimazaki, Jun ; Tsubota, Kazuo. / The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets. In: Investigative Ophthalmology and Visual Science. 2009 ; Vol. 50, No. 5. pp. 2109-2115.
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AU - Omoto, Masahiro

AU - Miyashita, Hideyuki

AU - Shimmura, Shigeto

AU - Higa, Kazunari

AU - Kawakita, Tetsuya

AU - Yoshida, Satoru

AU - Mcgrogan, Michael

AU - Shimazaki, Jun

AU - Tsubota, Kazuo

PY - 2009

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N2 - PURPOSE. To report the efficacy of human bone marrow-de- rived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. METHODS. Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in -modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithe- lial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air- liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cyto- keratin 3 (K3), K15, p63, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and trans- planted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochem- istry against K3 and K4. RESULTS. MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. CONCLUSIONS. MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

AB - PURPOSE. To report the efficacy of human bone marrow-de- rived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. METHODS. Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in -modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithe- lial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air- liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cyto- keratin 3 (K3), K15, p63, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and trans- planted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochem- istry against K3 and K4. RESULTS. MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. CONCLUSIONS. MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

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