ABSTRACT
Limbal stem cells (LSC) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell (HLECs) transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. A major challenge is the ability to identify LSC in vitro and in situ, and one of the major controversies in the field relates to reliable LSC markers. This study was carried out to evaluate the culture of a limbal biopsy on human amniotic membrane (HAM): directly on the chorionic side and on intact epithelium, and the expression of the stem cell associated markers: ABCG2, p63. HAM has been extensively used for ocular surface reconstruction and has properties which facilitate the growth of epithelial cells controlling inflammation and scarring.
Subject(s)
Amnion , Limbus Corneae/cytology , Limbus Corneae/growth & development , Stem Cells/cytology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , Cell Culture Techniques , Cornea/cytology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelium, Corneal/cytology , Epithelium, Corneal/metabolism , Feeder Cells , Humans , Limbus Corneae/metabolism , Membrane Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Stem Cells/metabolism , Tissue Culture TechniquesABSTRACT
INTRODUCTION: The human amniotic membrane is a highly abundant and readily available tissue that may be useful for regenerative medicine and cell therapy. AIM: To compare two previously published protocols for the isolation of human amnion mesenchymal stromal cells (hAMSCs), including their phenotypic characterization and in vitro potential for differentiation toward osteogenic, adipogenic, and chondrogenic mesodermal lineages. MATERIALS AND METHODS: Human placentas were obtained from selected caesarean-sectioned births. Two different protocols (Alviano et al. (1) and Soncini et al. (2) ) for the isolation of hAMSCs were performed. After monolayer expansion of adherent cells from both protocols, the cells were characterized by flow cytometry and for multipotentiality, as assessed by their capability to differentiate toward adipocyte-, osteoblast-, and chondrocyte-like cells. RESULTS: Both protocols yielded hAMSCs that showed plastic adherence, fibroblast-like growth, and well-defined human MSC markers. The cell yield and mesodermal differentiation capability of hAMSCs were higher in cells isolated using the Soncini protocol. CONCLUSIONS: Our data demonstrated the successful isolation of hAMSCs from full-term placentas using two published protocols. Differences between the two protocols in cell yield and in vitro differentiation potential are shown.
