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Maitee Urbieta, Enrique Salero, Victor L Perez; Isolation, characterization, and culture of human limbal stem cells from donor sclerocorneal rims. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3463.
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© ARVO (1962-2015); The Authors (2016-present)
The human cornea plays a role in light refraction, protection against toxicants, microorganisms and injury. Limbal stem cells (LSCs) are responsible for regenerating the corneal epithelium. Unlike differentiated corneal epithelium, LSCs exhibit a slow-cycling label retaining property and high proliferative potential and preferentially express DNp63α, ABCBG2, K15 and N-cadherin markers. The purpose of this work is the isolation, identification and characterization of human LSCs from donor sclerocorneal rims, in an effort to understand niche regulation, stem cell renewal and fate decisions in-vitro.
LSCs were obtained from direct dissection of human sclerocorneal tissue donors which ranged from 14 to 70 years of age. LSCs were isolated and cultured without feeder cells under stem cell conditions for 2-4 weeks. The resulting cultures were stained with rhodamine to evaluate colony-forming efficiency and maintenance of limbal epithelial stem/progenitor cell pool. Flow cytometry, western blotting and immunostaining were performed and total RNA was extracted from LSC at P0 and different passages (P1-P3) for gene expression assays.
The colony-forming efficiency of LSCs was considered optimal in all donor rims, regardless of age and gender, as evidenced by holoclone formation. Staining showed a higher intensity of the limbal stem cell markers DNp63α and K15 in LSC holoclones at P0 of culture, but not after passage/expansion of LSCs (P1-P2). These finding were consistent with flow cytometry analysis, which showed a higher expression of LSC markers in LSC colonies at P0, but decreased or absent expression of these markers after expansion. Similarly, gene expression and protein analysis revealed that cultured LSCs initially (P0) express all LSC markers, however, upon expansion, LSC marker expression decreases abruptly and corneal epithelium differentiation markers are turned on.
Human LSCs cultured in stem cells conditions maintain their ability to form colonies efficiently, however when these cells lose cell-cell contact during culture expansion, LSCs lose their stem cells properties and undergo differentiation. The work presented here highlights the potential application of freshly isolated LSCs in clinical regenerative medicine.
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