Human Amniotic Epithelial Cells as Novel Feeder Layers for ex vivo Expansion of Multipotent Limbal Epithelial Progenitor Cells

Abstract

Purpose:: Intact cryopreserved amniotic membrane with devitalized human amniotic epithelial cells (HAECs) can, but epithelially-denuded cryopreserved amniotic membrane cannot, help expand human limbal epithelial progenitor cells (HLECs) without murine 3T3 fibroblast feeder layers. We thus hypothesize that HAECs, a unique embryonic cell source, may be used as non-xenogenic feeder layers.

Methods:: HAECs were isolated from a fresh amnion by enzymatic digestion and serially cultured in different media varying in [Ca²⁺], FBS concentration, and supplements of different growth factors. Their culturing conditions as feeder cells were investigated by immunostaining to pan-cytokeratins(CK)/vimentin(Vim) and MTT assay. HLEC clonal cultures and subcultures in different media or feeder layers on mitomycin C-treated HAEC feeder layers were compared to those on traditional 3T3 fibroblast feeder layers regarding colony-forming efficiencies, differentiation and stem cell-associated markers.

Results:: HAECs uniformly expressed pan-CK and heterogeneously expressed Vim in vivo. These two subpopulations of Pan-CK(+)/Vim(+) and Pan-CK(+)/Vim(-) cells were maintained in serum-free media with high calcium, but some HAECs turned into pan-CK(-)/Vim(-) only in serum-free media with low or no calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media with an increase of proliferation, and could be subcultured for at least 8 passages in SHEM medium supplemented with EGF and insulin. Mitomycin C-treated HAEC feeder layers were significantly more effective in promoting clonal growth of HLEC progenitor cells than 3T3 feeder layers as judged by a smaller cell size, less K12 keratin expression, lack of connexin 43 expression, and higher percentages of stem cell-associated markers such as p63, Musashi-1 and ABCG2. Clonally expanded HLECs from HAEC feeder layers could further differentiate into neurons and nestin-positive neuronal progenitors when subcultured in serum-free, feeder cell-free KSFM medium or HAEC feeder layers, while those on 3T3 feeder layers might change into fibroblasts.

Conclusions:: Collectively, these results indicated that HAECs can be used as an alternative xeno-free feeder layer for ex vivo expansion of HLEC progenitor cells. Furthermore, this surrogate niche also help expand their fate decision to exhibit multipotency, an important property shared by a number of adult somatic stem cells, and may enhance their therapeutic potentials in the future.

Keywords: cornea: epithelium • plasticity • cornea: basic science