The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets.
OBJECTIVE
To report the efficacy of human bone marrow-derived 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 alpha-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 epithelial 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 cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted 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 immunohistochemistry 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, p63alpha, 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.
Omoto
Masahiro
M
Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Miyashita
Hideyuki
H
Shimmura
Shigeto
S
Higa
Kazunari
K
Kawakita
Tetsuya
T
Yoshida
Satoru
S
McGrogan
Michael
M
Shimazaki
Jun
J
Tsubota
Kazuo
K
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001854:Bone Marrow Cells; D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques; D017690:Cell Transplantation; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D016207:Cytokines / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology* / Q000637:transplantation; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D051523:Fibroblast Growth Factor 7 / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D006801:Humans; D053569:Keratin-15 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015703:Antigens, CD; C050361:CDH2 protein, human; C081876:CKAP4 protein, human; D015820:Cadherins; D016207:Cytokines; D053569:Keratin-15; D053558:Keratin-3; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D051523:Fibroblast Growth Factor 7; D017228:Hepatocyte Growth Factor