Neural potential of human limbal cells

Chen, Xiaoli, Hossain, Pawez, Griffiths, Helen, Scott, Jennifer and Lotery, Andrew J. (2015) Neural potential of human limbal cells. Investigative Ophthalmology & Visual Science, 56 (7), [3574].

Abstract

Purpose: Previous studies have shown that neural colonies (neurospheres) derived from adult mouse limbus are neural crest originated stem/progenitor cells, and can differentiate into functional neurons and/or cells expressing retinal specific markers in vitro. The aim of this study is to investigate whether limbal neurosphere cells (LNS) can be derived from aged human limbus, and differentiate into retinal like cells in vitro and in vivo.
Methods: Human limbal tissues used in this study were from two resources: (1) aged donor eyes from Bristol Eye Bank (age ranged from 72-97), and (2) healthy limbal tissue from live patients who underwent conjunctival surgery (size 1 mm2, age ranged from 34-85). Human limbus cells were isolated and cultured in the presence of mitogens. Following co-culture with developing retinal cells or in the presence of extrinsic factors, LNS and their progeny were characterized using immunocytochemistry and/or RT-PCR. Enhanced green fluorescent protein-tagged LNS were transplanted into the subretinal space of neonatal mice. The potential for limbal cells to differentiate into retinal like cells and integrate into the host retina was assessed by immunohistochemistry after 2-5 weeks.
Results: Human LNS were successfully generated from aged donor limbal tissues through a serum free sphere forming assay. Human LNS expressed neural stem cell markers, including Sox2 (31.2 ± 10.2%) and Nestin (34.8 ± 2.2%). For the superficial limbal tissues (1 mm2) obtained from live patients, no apparent LNS were generated, but cells expressing Nestin (3-5%) and early differentiated neuronal marker beta-III tubulin (15-20%) can be grown through explant culture in the presence of serum and mitogens. Following co-culture with developing retinal cells or in presence of extrinsic factors, low levels of retinal progenitor markers, such as Lhx2, Pax6 and Rx were detected in human LNS at the transcription level. Mature photoreceptor specific markers were not observed in human LNS either in vitro or in vivo.
Conclusions: Here we demonstrate that cells with neural potential can be derived from aged human limbal tissue or 1 mm2 of superficial limbal tissues from adult patients. Other approaches are needed to promote human limbal cells transdifferentiation into retinal lineage. However, their surgical accessibility and presence in aged individuals make them an attractive cell resource for autologous cell rescue of degenerative retinal diseases.

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Contributors

Author: Pawez Hossain

Author: Andrew J. Lotery

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