Human Amniotic Epithelial Cells as Novel Feeder Layers for Promoting Ex Vivo Expansion of Limbal Epithelial Progenitor Cells

摘要

Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK (+)/Vim(+) or pan-CK(+)/Vim(−) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK (+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least 8 passages especially supplemented with EGF and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43, but higher levels of stem cell-associated markers such as p63, Musashi-1 and ABCG2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus.