Human corneal stem cells display functional neuronal properties.
OBJECTIVE
Human corneal limbal stem cells mature and repopulate the superficial layers of the cornea throughout life. In this study, we tested the hypothesis that human corneal stem cells, derived from neural ectoderm, can exhibit functional neuronal properties.


METHODS
Human corneal limbal tissue (donor age 6 weeks to 92 years) was obtained from Upstate New York Transplant Services. Tissues were grown as explants on coverslips in DMEM with 10% calf serum. After 7-14 days in vitro, tissues on coverslips were double-immunostained for the stem cell marker, p63, along with nestin and neurotransmitter receptors GABA, dopamine, serotonin, glycine or acetylcholine. We also carried out whole cell current clamp and voltage clamp recordings on corneal stem cell cultures in order to determine their functional neurophysiological properties.


RESULTS
Co-localization of p63 with nestin, GABA receptor, glycine receptor, and serotonin receptor immunoreactivity was seen in a small number of cells in the corneal stem cell cultures. The resting potential of corneal stem cells was relatively low, approximately -13+/-8 mV (n=13; range -6 mV to -40 mV) measured in current clamp. No action potentials or voltage sensitive Na+ and K+ currents were detected. However, in a small number of cells, kainic acid (0.5 mM), a non-NMDA glutamate receptor agonist, and GABA induced a small inward current. Glutamate receptor antagonist, CNQX, and GABA receptor antagonist, bicuculline and CGP-35348 blocked the agonist response.


CONCLUSIONS
A subpopulation of human corneal stem cells exhibit neuronal properties in vitro, as evidenced by immunoreactivity to nestin, GABA receptor, glycine receptor, and serotonin receptor, as well as functional neurophysiological responses to GABA and kainic acid. Human corneal stem cells may represent a potential source of non-embryonic, autologous, surgically-accessible graft material with neuronal potential.
Seigel
Gail M
GM
Department of Ophthalmology, University at Buffalo, Buffalo, NY 14214, USA. gseigel@frontiernet.net
Sun
Wei
W
Salvi
Richard
R
Campbell
Lorrie M
LM
Sullivan
Susan
S
Reidy
James J
JJ
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000032:analysis*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004268:DNA-Binding Proteins; D004475:Ectoderm / Q000502:physiology; D004594:Electrophysiology; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D016147:Genes, Tumor Suppressor; D006801:Humans; D007124:Immunoenzyme Techniques; D007223:Infant; D007381:Intermediate Filament Proteins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000502:physiology; D008565:Membrane Proteins; D008875:Middle Aged; D009419:Nerve Tissue Proteins; D064231:Nestin; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010750:Phosphoproteins / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000037:antagonists & inhibitors / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins
D015415:Biomarkers; C081876:CKAP4 protein, human; D004268:DNA-Binding Proteins; D007381:Intermediate Filament Proteins; D008565:Membrane Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D010750:Phosphoproteins; D017981:Receptors, Neurotransmitter; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins