Adult ciliary epithelial stem cells generate functional neurons and differentiate into both early and late born retinal neurons under non-cell autonomous influences
© Del Debbio et al.; licensee BioMed Central Ltd. 2013
Received: 27 June 2013
Accepted: 14 October 2013
Published: 22 October 2013
The neural stem cells discovered in the adult ciliary epithelium (CE) in higher vertebrates have emerged as an accessible source of retinal progenitors; these cells can self-renew and possess retinal potential. However, recent studies have cast doubt as to whether these cells could generate functional neurons and differentiate along the retinal lineage. Here, we have systematically examined the pan neural and retinal potential of CE stem cells.
Molecular and cellular analysis was carried out to examine the plasticity of CE stem cells, obtained from mice expressing green fluorescent protein (GFP) under the influence of the promoter of the rod photoreceptor-specific gene, Nrl, using the neurospheres assay. Differentiation was induced by specific culture conditions and evaluated by both transcripts and protein levels of lineage-specific regulators and markers. Temporal pattern of their levels were examined to determine the expression of genes and proteins underlying the regulatory hierarchy of cells specific differentiation in vitro. Functional attributes of differentiation were examined by the presence of current profiles and pharmacological mobilization of intracellular calcium using whole cell recordings and Fura-based calcium imaging, respectively. We demonstrate that stem cells in adult CE not only have the capacity to generate functional neurons, acquiring the expression of sodium and potassium channels, but also respond to specific cues in culture and preferentially differentiate along the lineages of retinal ganglion cells (RGCs) and rod photoreceptors, the early and late born retinal neurons, respectively. The retinal differentiation of CE stem cells was characterized by the temporal acquisition of the expression of the regulators of RGCs and rod photoreceptors, followed by the display of cell type-specific mature markers and mobilization of intracellular calcium.
Our study demonstrates the bonafide retinal potential of adult CE stem cells and suggests that their plasticity could be harnessed for clinical purposes once barriers associated with any lineage conversion, i.e., low efficiency and fidelity is overcome through the identification of conducive culture conditions.
KeywordsStem cells Ciliary epithelium Photoreceptors Retinal ganglion cells Cell therapy Retina
More than a decade ago two labs independently discovered that the adult rodent CE, a tissue of neuroectodermal origin between the retina and retinal pigment epithelium (RPE), contained a small subset of cells, which displayed neural stem cell properties in vitro[1, 2]. These cells, when removed from their niche and cultured in the presence of mitogens proliferated and generated clonal neurospheres. Cells in adult CE neurospheres were proliferative, expressed pan-neural and retinal progenitor markers and differentiated along pan-neural and retinal lineages [1–5]. Further characterization revealed that these are a rare population of adult CE cells and unlike progenitors in the embryonic retina they displayed a cardinal feature of stem cells, i.e., they could self-renew [1, 3]. The presence of such cells in rodent eyes was re-confirmed [6–11] and the evidence for their presence in postnatal chicken , rabbit , porcine [14, 15], humans [9, 16–18] and monkeys  emerged, suggesting evolutionary conservation of such cell population in adult vertebrate eyes. Further examination of their properties in rodents showed their relationship with retinal progenitor cells at the transcriptome level [8, 19]. The conservation of adult CE stem cells in higher vertebrates, their retinal progenitor properties and ability to differentiate along retinal lineage in vitro[1, 2, 4, 13, 16, 20] and in vivo[20, 21] suggested that these cells may be analogous to regenerative cells in the ciliary margin zone of the lower vertebrates [22–24]. Thus, these relatively accessible adult stem cells were posited as an alternate heterologous source from which progenitors with retinal potential may be derived for retinal cell therapy . However, two recent reports suggested that adult CE stem cells do not possess neural or retinal potential. Cicero et al., 2009 , based on the pigmented and epithelial features of these cells, questioned their characterization as retinal stem cells and reported that they lack the potential to differentiate along bonafide neural and retinal lineages. Gualdoni et al., 2010 , using Nrl-GFP mice , for genetic labeling of adult CE cells, enabling their lineage tracing along the rod photoreceptor lineage, and monolayer adherent culture concluded that these cells fail to differentiate into rod photoreceptors. Consequently, we have determined whether or not adult CE stem cells possess the capacity for pan-neuronal and retinal differentiation by systematically examining the temporal acquisition of the expression of cell-type specific regulators and phenotype specific markers along with the display of cell-type specific functional attributes. Our study not only confirms that these cells generate functional neurons but also demonstrates that like retinal progenitors they respond to specific culture conditions simulating the environment during retinal histogenesis and differentiate into both early and late born retinal neurons with functional attributes. Thus our study demonstrates that the adult CE stem cells do possess retinal potential and suggests that their plasticity could be harnessed for potential clinical purposes once the barriers associated with lineage conversion, i.e., low efficiency and fidelity, are overcome through the identification of conducive culture conditions.
The growth factor-responsive cells in adult CE  may represent the evolutionarily conserved counterparts in the ciliary margin zone that sustains regeneration in the lower vertebrates [24, 46]. Though the physiological role of these cells remains obscure their presence and accessibility offers an opportunity for their use in regenerative medicine, given their self-renewing capacity and plasticity in vitro. Based on their location and stem cell properties that they display in vitro they were termed adult CE stem cells [2, 22]. Alternatively, based on their progenitor properties and ability to differentiate along retinal lineage in vitro, they were characterized as retinal stem cells , the existence of which was questioned by Cicero et al., 2009  on the grounds of their pigmentation, epithelial features, and low efficiency of retinal differentiation. Pigmentation was recognized early on as a feature of the epithelium to which adult CE stem cells belonged and their retinal potential was explained based on their reprogramming in vitro[2, 22]. Such reprogramming, at the light microscopic level, was suggested by the observation that some adult CE stem cells lose their pigmentation as they divided in vitro, and that the majority of cells that displayed differentiated phenotypes were apparently devoid of pigmentation [1, 2]. In contrast, ultra-structure analyses suggested that the reprogramming might not lead to complete erasure of parental properties as these cells maintained some pigmentation and epithelial features [7, 9]. The pigmentation and epithelial features were regarded as contradictory to the nature of stem cells with retinal potential . However, pigmentation as an exclusionary criterion demands caution. For example, metabolic products such as melanin, which are resistant to degradation , may not be a reliable indicator of the lack of lineage conversion as they may persist long after the expression of genes associated with their biosynthesis has been attenuated. Also, the presence of stem cell properties and epithelial features such as the presence of adherence junctions, tight junctions, and gap junctions are not mutually exclusive; neuroepithelium and their stem cell derivatives possess these features and their role in the regulation of stem cells, both embryonic and somatic, has begun to emerge [4, 48–50]. The presence of morphological features of differentiated cells such as microvilli may not be an exclusionary criterion  either, given the evidence that some stem cells, embryonic [51, 52] and somatic [53, 54] display such features. In the adult retina a subset of highly morphologically and functionally differentiated cells, Müller glia, possess stem cell properties and undergo re-programming to sustain regeneration [46, 55].
The low efficiency of differentiation along the retinal lineage is not unexpected where reprogramming of heterologous cells are required. If trans-differentiation is invoked, where a post-mitotic adult CE cell converts into a post-mitotic retinal cell without an intermediate step, the built-in stochasticity plus the lack of optimal differentiation conditions will predict a low lineage conversion. If de-differentiation is considered, where a small subset of cells first reverts to a proliferative and developmentally immature stage before differentiating along a particular lineage, their relative enrichment in particular culture conditions will be reflected in the conversion efficiency. In either case, culture conditions will play a critical role in the outcome of the lineage conversion and that may be an explanation for the results of Cicero et al., 2009  and Gualdoni et al. 2010 . It is quite likely that the relatively high efficiency of retinal conversion reported recently by Demontis et al., 2012  is likely to their culture conditions that favored the enrichment of adult CE stem cells. Given the fact that adult CE stem cells are derived from the same embryonic neuroepithelium as retina and their counterparts in lower vertebrates sustain the growth of the retina in adults  their differentiation along retinal lineage is not unexpected. Cells from another derivative of ocular neuroepithelium, RPE, possess the capacity to differentiate into retinal neurons in vitro, however, this plasticity is limited to embryonic stages . Inter-conversion of cell types within the same lineage is not uncommon. Pancreatic and hepatic cells share the same embryonic lineage and trans-differentiation of hepatic cells into pancreatic cells and vice versa is well documented . Inherent in the use of heterologous stem cells, including cells with pluripotent potential, is the barrier of low efficiency of lineage conversion. The challenge for these cells, including adult CE stem cells, is to increase the efficiency, efficacy, and fidelity of differentiation into specific retinal cell types. If that were achieved, their usefulness for retinal cell therapy would not be in doubt.
In summary, we have demonstrated that adult mouse adult CE stem cells, isolated by neurosphere culture, undergo reprogramming that attenuates the expression of select adult CE-specific genes and acquire the expression of those that characterize retinal progenitors. A subset of these reprogrammed adult CE stem cells display potential to respond to environmental cues, specific to early and late stages of retinal histogenesis and give rise to RGCs and rod photoreceptors with functional attributes. They also generate functional neurons under non-cell autonomous influence. The wide variation of the efficiency of photoreceptor differentiation of adult CE stem cells or lack thereof is likely due to different culture conditions used in different labs. Indeed, the presence of melanin and epithelial features points toward the property of the epithelium to which these cells belong, but do not exclude their characterization as stem cells, when they display multipotetiality and self-renewal in vitro. The potential of adult CE stem cells to differentiate into both early and late born retinal neurons suggests their usefulness for cell therapy for retinal degeneration once the efficiency of non-cell autonomous re-programming is reproducibly increased.
Animals, CE cells and neurospheres assay
This study was ethically approved by the Institutional Animal Care and Use Committee (IACUC), at University of Nebraska Medical Center and Nrl-GFP mice, a gift from Dr. Anand Swaroop , were housed and bred in the Department of Comparative Medicine at University of Nebraska Medical Center. Isolation and culture of adult CE cells from adult Nrl-GFP mice  (6-8 weeks old) was performed as previously described [2, 3, 19]. Briefly, after eye enucleation, cornea, lens, iris and retina were removed to eliminate any potentially dividing cells from these tissues as contaminants. The pigmented CE was incubated in HBSS, containing collagenase (78 U/ml, Sigma) and hyaluronidase (38 U/ml, Sigma) for 35 min at 37°C, followed by dissociation in 0.25% trypsin, 1 mM EDTA, and 20 mg/ml DNase1 for another 35 min. The presence and absence of Tyrosinase (Figure 1) and rod photoreceptor specific transcripts (Figure 3), respectively, by PCR reflected the purity of CE cell dissociates. CE cells were cultured in retinal culture medium (RCM)  containing FGF2 (10 ng/ml, R&D Systems) and EGF (20 ng/ml, R&D Systems) for 6 days to generate the CE neurospheres.
RGC and rod photoreceptor differentiation
For RGC differentiation, primary neurospheres were cultured in CM collected from the culture of embryonic day 14 rat retinas (=E14CM), diluted in RCM (1:1) . For rod photoreceptor differentiation, primary neurospheres were cultured on poly-d-lysine and laminin coated glass coverslip in conditioned medium (CM) collected from the culture of postnatal day 1 rat retina (=PN1CM), diluted in RCM (1:1) , supplemented with1% B27 (Invitrogen), DAPT (3 μM; Sigma), Sonic hedgehog (Shh, 3 nM; R&D Systems), Taurine (100 μM; Sigma), all-trans Retinoic acid (500 nM; Sigma), and 2% KOSR (Knockout serum replacement, Gibco). CM was centrifuged and filtered before use to eliminate the possibility of cell contamination.
Polymerase chain reaction
List of specific primers
F: 5′- ATTCTCTTCCTCTCTCCCTGCTGC -3′
R: 5′- GCTACCATAAATCAAGGTGCGTCC-3′
F: 5′- AGCCAGCAGAAAAGAAATGTGC-3′
R: 5′- ATTGGGGTCAGGGGAGAAAGAC-3′
F: 5′- CGAGCGTGTGGTCATCAACATC-3′
R: 5′- CATTGCGGAGTGGGTCAAAG-3′
F: 5′- CATCAAGGAAGAGGAGAAGCCC -3′
R: 5′- GAGAATGACCAAGACCGACACG -3′
R:5′- CACGCATCACGAAGTCGTTC -3′
F:5′- ACCAAGCCAGATGCCTGAAA -3′
R: 5′- CAACACCTTCCTTGGCAATGG-3′
R: 5′- GGAAAGGCAGGTTGGAAAACAC-3′
F: 5′-ACAGTCCATGCCATCACTGCC -3′
List of primary antibodies
Affinity Bioreagents (554895)
Santa Cruz (sc-79031)
BD Pharmingen (554895)
The protein samples (50 μg), extracted from cultured cells using T-PER Tissue Protein Extraction Reagent (Thermo Scientific), were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel (12%) electrophoresis, and then transferred onto the 0.45 micron PVDF-Plus Transfer Membrane (GE Water & Process Technologies). Membranes were blocked in TBS-Tween (25 mm Tris-HCL, pH 8.2, 144 mm NaCl, 0.1% Tween-20), with 5% skim milk and 1% BSA for 2 hours at room temperature, followed by incubation with primary antibodies (Table 2) overnight, at 4°C. Membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, and visualized with an enhanced chemiluminescence reagent (ECL Plus Western Blotting Detection System, Amersham).
Cells were dissociated by trypsinization (0.25%) for 10 min, washed with 1X PBS, and fixed with 4% paraformaldehyde for 15 minutes, followed by permeablization in 0.2% saponin, containing 5% NGS, for 30 min. Cells were incubated, first with respective primary antibodies (Table 2) for 2 hours and washed with PBS containing 0.2% saponin and then with secondary antibody conjugated to Cy5 for 1 hour, followed by analysis on an LSRII Flow Cytometry System (BD Biosciences).
Differentiated cells were incubated in culture medium containing acetoxy-methyl ester Fura-2 (5 μM, Invitrogen) for 30 min at 37°C. After a 15-minute rinse in culture medium without Fura-2 AM the coverslip was mounted on the perfusion chamber, fitted on an inverted Olympus microscope (IX70). Test solutions [20 mM (S)-3,4-dicarboxyphenylglycine (DCPG); 100 mM (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), and 200 nM N-methyl-D-aspartate (NMDA), Tocris,) were applied by bath perfusion. Cells were subjected to excitation wavelength at 340 and 380 nm, using Lamda DG-4 (Sutter Instrument). Fluorescence changes were monitored every 5 seconds by cooled charge-coupled device (CCD) camera (Orka II, Hamamatsu) and ratio-metric imaging carried out using Open Lab Software (Improvision).
Membrane currents were recorded under whole-cell patch configuration . The cells were voltage clamped at the steady membrane potential of -80 mV and currents were induced by voltage steps (-80 mV to -50 mV in the first step, then to +20 mV in increments of 10 mV). The bath (extracellular) solution contained (mM): NaCl, 160; KCl, 4.5; CaCl2, 2; MgCl2, 1; HEPES, 5; glucose, 11; adjusted to pH 7.3 with NaOH. The pipette solution contained (mM): KCl, 150; CaCl2, 1; MgCl2, 2; EDTA, 11; HEPES, 10; adjusted to pH 7.3 with KOH. Experiments were performed at room temperature. The patch pitettes of 2-5 MΩ were fabricated on a two-stage puller (PC-10, Narishige). Currents were amplified using an Axopatch 200B amplifier (Axon instruments), filtered at 1 kHz, digitized at 5 kHz using a digital 1440A digitizer, and recorded using pClamp10 software. Junction potentials were corrected and the cell capacitance was compensated (~70%) in all cells tested.
Cell type-specific antigen-positive cells were counted in 10-15 randomly selected fields in three to five different coverslips. Each experiment was repeated at least three times. Values were expressed as ± SEM. Data were analyzed using the Student’s t-test or ANOVA to determine the significance of the differences between treatment and control groups.
Retinal ganglion cells
Retinal pigmented epithelium
Retinal culture medium
Postnatal day 1 conditioned medium
Embryonic day 14 conditioned medium
Green fluorescence protein.
Thanks are due to Dr. Sowmya Parameswaran, Ramkishore Gernapudi, Ani Das, Chandrika Abouri and Graham Sharp for help in the CE stem cells project and critical reading of the manuscript. This work was supported by Lincy Foundation, Pearson Foundation, and Otis Glebe Foundation.
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