Effects of different extracellular matrices and co-cultures on human limbal stem cell expansion in vitro.
To elucidate the effect of extracellular matrices (ECMs) and related and nonrelated-limbal feeder cells as substitutes for the in vivo niche on the phenotype and genotype of the limbal stem cell (SC) expansion in vitro, human limbal SCs were used. The limbus explants were expanded on human amniotic membrane (AM), commercial ECMs including matrigel (MAT), collagen (COL), and control (no ECM) in presence and absence of feeder cells including human limbal fibroblasts (LFs), a limbus-specific cell and mouse embryonic fibroblasts (MEFs). Proliferation, cell death, immunocytochemistry, expression of specific genes, ultrastructural characteristics, and size and granularity of expanded human limbal SCs in different groups were evaluated. The growth, cell proliferation, and survival of limbal SCs were enhanced by AM and MAT matrices. Ultrastructure and expression of stemness markers revealed that there was no significance difference between AM and MAT. However, flow cytometric analysis showed that the size and granularity of cultured cells increased in the presence of MAT and COL as well as in no ECM group. Moreover, co-culturing of limbal explants with LFs and MEFs on AM and MAT groups, enhanced the expansion and survival of cultured cells in comparison with others. In conclusion, the cultivation of human limbal explants on AM co-culturing with human LFs promises to be a good model for preparing undifferentiated epithelial sheets suitable for transplantation.
Ahmadiankia
Naghmeh
N
Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Ebrahimi
Marzieh
M
Hosseini
Ahmad
A
Baharvand
Hossein
H
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000328:Adult; D000650:Amnion / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002102:Cadaver; D018920:Coculture Techniques / Q000379:methods; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C081876:CKAP4 protein, human; D004338:Drug Combinations; D007797:Laminin; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen