Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation.
To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTRimmunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in β-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear β-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/β-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes.
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.
Yi-Jen
YJ
Hsueh
Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Shiang-Fu
SF
Huang
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.|Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan.|Biomedical Engineering Research Center, Chang Gung University, Taoyuan, Taiwan.
Jui-Yang
JY
Lai
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.
Shih-Chieh
SC
Ma
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.|Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Hung-Chi
HC
Chen
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.
Sung-En
SE
Wu
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.
Tze-Kai
TK
Wang
Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan.|Department of Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Chi-Chin
CC
Sun
Department of Dentistry, College of Oral Medicine, Chung Shan Medical University, Taichung, Taiwan.
Kevin Sheng-Kai
KS
Ma
Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.|Department of Physiology, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Jan-Kan
JK
Chen
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou, Taiwan.
Chyong-Huey
CH
Lai
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan.|Center for Tissue Engineering, Chang Gung Memorial Hospital, Linkou, Taiwan.|Department of Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
David Hui-Kang
DH
Ma
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D055815:Young Adult; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism; D000099200:Scaffold Protein ILK
D017364:Collagenases; D017346:Protein Serine-Threonine Kinases; D051176:beta Catenin; D000099200:Scaffold Protein ILK