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TISSUE-SPECIFIC STEM CELLS

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

^aTissueTech, Inc., and Ocular Surface Center, Miami, Florida, USA;
^bDepartment of Ophthalmology and
^cInstitute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan

Key Words. ATP-binding cassette subfamily G2 • Amniotic membrane • Amniotic epithelial cells • Ex vivo expansion • Feeder layers Connexin 43 • Cytokeratins K12 • Limbus • Musashi-1 • Neuronal progenitors • Plasticity • Progenitor cells • p63 Stem cells

Correspondence: Scheffer C. G. Tseng, M.D., Ph.D., Ocular Surface Center, 7000 SW 97 Avenue, Suite 213, Miami, Florida 33173, USA. Telephone: 305-274-1299; Fax: 305-274-1297; e-mail: stseng{at}ocularsurface.com

Received October 25, 2006; accepted for publication May 4, 2007.
First published online in STEM CELLS EXPRESS   May 10, 2007.



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 eight passages, especially supplemented with epidermal growth factor 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 ATP-binding cassette subfamily G2 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.

Disclosure of potential conflicts of interest is found at the end of this article.

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