Abstract
BackgroundThe 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.ResultsMolecular 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.ConclusionsOur 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.
Highlights
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
The proportion of cells expressing Rx and Pax6 immunoreactivities was 63 ± 0.65% and 56 ± 0.52%, respectively. These observations suggested that cells that generated CE neurospheres displayed the properties of their resident epithelium, which were progressively attenuated as retinal progenitors markers were temporally expressed in vitro
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
Summary
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. 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 [6,7,8,9,10,11] and the evidence for their presence in postnatal chicken [12], rabbit [13], porcine [14,15], humans [9,16,17,18] and monkeys [18] emerged, suggesting evolutionary conservation of such cell population in adult vertebrate eyes. 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
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