|Molecular Vision 2003;
Received 16 December 2002 | Accepted 28 April 2003 | Published 30 April 2003
Human corneal stem cells display functional neuronal properties
Gail M. Seigel,1,2
Lorrie M. Campbell,1,2
James J. Reidy1
Departments of 1Ophthalmology and 2Physiology & Biophysics, University at Buffalo, Buffalo, NY; 3Center for Hearing & Deafness, University at Buffalo, Buffalo, NY; 4Upstate New York Transplant Services, Buffalo, NY
Correspondence to: Gail M. Seigel, Departments of Ophthalmology and Physiology & Biophysics, University at Buffalo, Sherman 124, 3435 Main Street, Buffalo, NY, 14214; Phone: (716) 829-2157; FAX: (716) 829-2344; email: firstname.lastname@example.org
Purpose: 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.
Stem cells are responsible for cellular replacement and tissue regeneration throughout life, yet the location(s) of all adult stem cells and the destination(s) to which their progeny migrate is limited in adults. Stem cells in the corneoscleral limbus [1,2] are surgically accessible and participate in the dynamic equilibrium of the corneal surface, replacing superficial epithelial cells that are shed and sloughed off during eye-blinking . Despite their non-neuronal location, corneal stem cells are derived from neural ectoderm, from which the central nervous system originates . Therefore, we tested the hypothesis that human corneal stem cells exhibit functional neuronal properties characteristic of their neuroectodermal origin.
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, cells on coverslips were double-immunostained for the corneal stem cell marker p63, along with neurotransmitter receptors GABA, dopamine, serotonin, glycine or acetylcholine, as well as the neuronal stem cell marker nestin. HTERT staining was also used to detect the presence of telomerase as an indication of proliferation.
The following primary antibodies were used: mouse anti-GABA A receptor B-chain (Research Diagnostics, Flanders, NJ), rabbit anti-serotonin receptor (Santa Cruz Biologicals, Santa Cruz, CA), mouse anti-acetylcholine receptor B (BD Transduction Laboratories, San Diego, CA), rabbit anti-glycine receptor (Chemicon Int., Temecula, CA), rabbit anti-dopamine receptor (used as an isotype control for rabbit antibodies; Chemicon), mouse anti-p63 (BD Pharmingen, San Diego, CA), mouse anti-nestin (Chemicon), and rabbit anti-hTERT (Calbiochem, San Diego, CA)
Cells were fixed in 10% paraformaldehyde for 10 min. After a rinse in phosphate-buffered saline (PBS), cells on coverslips were incubated for one h with primary antibody. After rinsing 3 x 5 min in PBS, sections were incubated with a 1:1500 dilution of biotinylated goat anti-rabbit or anti-mouse immunoglobulin (Vector Laboratories, Burlingame, CA) for 60 min. Tissue sections were incubated for 20 min with horseradish peroxidase-conjugated avidin (Elite kit, Vector Laboratories). The sections were rinsed in 0.05 M Tris and antigens were detected with diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA); the brown/black reaction product was visualized by light microscopy. Negative controls consisted of incubations in control 5% goat serum or rabbit anti-dopamine as the primary antibody, and did not generate reaction product. For double-immunostaining, cells underwent the same protocol the second day, with mouse anti-p63 as the primary antibody. Instead of DAB substrate, the cells were incubated with red AEC substrate (Vector Laboratories) for 10 min. For hTERT, AEC substrate was used. After two rinses in water, the cells on coverslips were mounted onto slides with Mowiol (Calbiochem).
Prior to recording from corneal stem cells, the culture medium was replaced with Hank's Balanced Salts Solution which contained: 137 mM NaCl, 0.2 mM Na2HPO4, 5.4 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO2, 1.3 mM CaCl2, 5.6 mM glucose, and 10 mM HEPES. The recording pipette was filled with a solution containing: 120 mM KCl, 20 mM KF, 2 mM NaCl, 2 mM MgCl2-2H2O, 10 mM EGTA, and 10 mM HEPES. pH was set to 7.3 with KOH and the osmolarity was adjusted with sucrose to 290 mOsm. The whole-cell recording configuration was established on the soma of cells (1-3 GW) and recordings were made at room temperature (about 22 °C). Current and voltage signals were amplified using a patch-clamp amplifier (Axon 700A) and digitized (DigiData 1200, Axon Inst.) and analyzed by pCLAMP (8.01 Axon Inst.). Kainic acid (KA), γ-aminobutyric acid (GABA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), bicuculline (BIC, GABAa receptor antagonist) and CGP-35348 (GABAb receptor antagonist), were all purchased from Sigma company. The solutions were made in HBSS and applied alone or mixed together through a puffer electrode connected to a DAD-12 super fusion system (ALA Inst.).
Stem cells derived from human corneal limbal tissue exhibited a flat, non-neuronal, epithelioid morphology under standard culture conditions. Cells were stained with the corneal stem cell marker p63 , along with the neuroepithelial marker nestin , to determine the presence of this stem cell intermediate filament protein. As seen in Figure 1A, a few cells appeared to co-localize p63 and nestin (long arrow). Many cells expressed nuclear p63 without nestin immunoreactivity (Figure 1A, small arrow). At the periphery of cellular outgrowth, we observed a significant number of nestin positive, p63-negative cells (Figure 1B).
hTERT is a telomerase subunit expressed in proliferative cells that have escaped senescence . We immunostained our stem cell cultures to detect the presence of hTERT as another way of demonstrating their proliferative nature, in addition to p63 immunoreactivity. hTERT immunoreactivity was detected in a few cells within our cultures. One example of an hTERT-immunopositive cell is shown inFigure 2.
To assess inherent neuronal properties, cells were double-immunolabeled for neurotransmitter receptors and p63. Co-localization of the nuclear p63 stem cell marker with GABA receptor, glycine receptor, and serotonin receptor immunoreactivity was seen in a small number of cells in corneal stem cell cultures. Neurotransmitter receptor immunolabeling appeared either as brown ridges or as a "buck-shot" pattern coupled with a red p63-immunoreactive nucleus (Figure 3). No immunoreactivity was seen for acetylcholine or dopamine receptors. The rabbit anti-dopamine antibody was used as an isotype control (Figure 3D).
To determine if functional neurotransmitter receptors were present, we made whole-cell voltage clamp and current clamp recordings from corneal stem cells (Figure 4). The resting potential of human corneal stem cells varied from cell-to-cell, but the average 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 (3 of 17), kainic acid (0.5 mM), a non-NMDA glutamate receptor agonist, and/or GABA (1 mM), induced a small inward current. The glutamate receptor antagonist, CNQX (0.1 mM), and the GABA receptor antagonists, bicuculline (0.5 mM) combined with and CGP-35348 (0.5 mM), blocked the agonist response.
In this study, we show that a subpopulation of human corneal stem cells exhibit functional neuronal properties in vitro, as evidenced by immunoreactivity to nestin, GABA receptor, glycine receptor, and serotonin receptor, as well as functional electrophysiological responses to GABA and kainic acid. Recent reports from a number of investigators demonstrate that stem cells derived from non-neuronal tissues can generate cells with neuronal phenotypes. Stem cells isolated from muscle , bone marrow , and even dental pulp  can be induced to display neuronal characteristics. Initial evidence for the possibility of neuronal characteristics in corneal limbal cells was recently shown in rodent, in which dissociated corneal limbal epithelial cells responded to epidermal growth factor and fibroblast growth factor-2 by expressing neuronal markers, such as nestin and Notch-1 . Here, we show that a subpopulation of human corneal stem cells express functional neuronal markers without the influence of differentiating treatments. An important contribution of the current study is the demonstration that human corneal stem cells express several important neurotransmitter receptors that can be activated by exogenously applied neurotransmitters. These receptors could conceivably guide the differentiation and integration of these stem cells.
There are some important caveats to our findings. The corneal limbal tissue harvested for our cultures may contain contaminating cell types, including epithelial cells and transient amplifying cells (TA cells). Challenges remain in the iron-clad identification of corneal stem cells in this system. The p63 marker is the best identification tool that has been demonstrated to distinguish corneal stem cells from their TA progeny . However, it is possible that some types of contaminating epithelial cells may utilize neurotransmitter receptors for non-neuronal purposes, as corneal epithelium has been shown to express specific serotinergic receptors . However, it is unlikely that these same epithelial cells would co-express the stem cell marker p63 or nestin. One additional stem cell identification tool, not suitable for human studies, is the retention of BrdU labeling as an indicator of proliferation. In lieu of BrdU staining, we chose hTERT as another indicator of cell proliferation. Further studies will benefit from the development of additional definitive corneal stem cell markers
Human corneal stem cells may represent a potential source of non-embryonic, autologous, surgically-accessible graft material with neuronal potential. Human corneal stem cells, with a life-long capacity to mature and repopulate the surface of the cornea, are already being used as graft material in human patients with corneal stem cell deficiencies [13,14]. A healthy eye can tolerate a partial limbal excision of one by two mm . Once excised, autologous limbal tissue can then be propogated in vitro  and transplanted back into the recipient without the need for systemic immunosuppression. One would anticipate that the differentiation potential of corneal stem cells is dependent upon a myriad of external as well as internal cues. Further treatments with neurotrophic factors may enhance the differentiation of human corneal stem cells toward neuronal phenotypes, with the ultimate goal of replacing lost/damaged neurons of the central nervous system.
The authors thank Jean Kasperek for technical assistance. Funding provided by NIH grant P01 DC03600-01A1, Upstate New York Transplant Services, and the Western New York Lions Blind and Charity Fund.
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