The potential of limbal neurosphere cells to differentiate into retinal cell phenotypes

Chen, Xiaoli, Thomson, Heather, Hossain, Parwez and Lotery, Andrew (2013) The potential of limbal neurosphere cells to differentiate into retinal cell phenotypes. Investigative Ophthalmology & Visual Science, 54 (15), 2231.

Abstract

Purpose: Neural colonies (neurospheres) can be generated from adult corneal limbus in vitro. We previously showed that neurospheres derived from limbus were of neural crest origin and can differentiate into functional neurons in vitro. The aim of this study was to investigate whether limbal neurosphere cells (LNS) could differentiate into retinal like cells in vitro and in vivo when exposed to a retinal developing microenvironment.
Methods: Cells from adult (8-week old) murine corneal limbus were isolated and cultured in a serum-free sphere-forming system in the presence of mitogens. Following co-culture with developing retinal cells in vitro, LNS and their progeny were characterized using immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). Enhanced Green Fluorescent Protein (eGFP) labelled limbal neurosphere cells were transplanted into the subretinal space (SRS) 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: LNS cells formed a monolayer, with cells displaying neuronal morphologies following co-culture with neonatal retinal cells. Expression of retinal progenitor cell markers Pax6, Lhx2 and Six6 were observed by RT-PCR after 2-4 days of co-culture. Following 7-10 days in co-culture, LNS cells were immunopositive for the photoreceptor specific marker rhodopsin (13 ± 3%), the mature neuronal marker neurofilament 200 (31 ± 10%) and a major component of synapses within the retina Syntaxin3 (9± 2%)). LNS also displayed ultrastructural changes following differentiation. TEM revealed loss of junctions within the LNS and the presence of putative presynaptic dense bodies. In addition, non-motile primary cilia were also detected in differentiated LNS cells. Although not specific to retinal lineage cells, the same subtype of sensory cilia is present in photoreceptor and RPE cells. Following transplantation into the SRS of wildtype neonatal mice, rhodopsin was detected in eGFP labelled LNS cells.
Conclusions: These data highlight that corneal limbal stromal stem/ progenitor cells can transdifferentiate to a retinal like phenotype in vitro and in vio. Therefore LNS cells could be a potential resource for autologous cell rescue of degenerative retinal diseases.

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Contributors

Author: Parwez Hossain

Author: Andrew Lotery

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