PMC

Isolation of stem cells from the human ciliary epithelium and neural retina. (A) The retina and ciliary body were detached from the choroid and RPE. (B) Pigmented cells were gently brushed away from the ciliary epithelium. (C) Microscopic appearance of pigmented CE cells upon isolation. (D) Broken line indicates the site from which non-pigmented ciliary epithelium was cut away from the neural retina. (E) Microscopic appearance of non-pigmented ciliary epithelial cells immediately after isolation. (F) Microscopic appearance of cells obtained from the neural retina immediately after isolation. (CE = ciliary epithelium, NPE = non-pigmented ciliary epithelium, NR = neural retina. Scale bars represent 20 μM.)

Phase-contrast microscopy of cells isolated from the neural retina and CE cultured in suspension for 1 week before transferal to adherent culture conditions (A) Cells isolated from the neural retina did not form spheres when cultured in suspension. (B) Neural retinal cells were able to adhere to tissue culture plates after initial culture in suspension for 1 week. However, these cells did not proliferate. (C) Pigmented CE cells did not adhere after 72 h in suspension culture. (D) When transferred to adherent conditions, pigmented CE cells attach, lost their pigment and acquired a large flattened/epithelioid morphology and did not proliferate. (E) Non-pigmented CE cells formed spheres after 72 h in suspension culture. (F) When cultured under adherent conditions in the presence of EGF, dissociated CE cells from non-pigmented spheres reached confluency by 1 week and displayed a characteristic epithelial morphology. (Scale bars represent 100 μm. CE = Ciliary Epithelium, EGF = Epidermal Growth Factor.)

Expression of stem cell markers by Müller glia and non-pigmented CE (A) RT-PCR analysis of two Müller stem cell lines that became spontaneously immortal (MIO-M1 and MIO-M7) and two non-pigmented CE cell preparations (CE 6165 and CE 6213). All cells expressed the retinal progenitor markers SHH, PAX6, CHX10, SOX2 and NOTCH1. However, a lower expression of SOX2 was observed in non-pigmented CE cells, when compared with Müller stem cells. GAPDH primers were used as control. (B) Immunostaining of Müller and non-pigmented CE cells for markers of retinal progenitors. A proportion of Müller stem cells showed expression of CHX10, SHH and SOX2, whilst non-pigmented CE cells did not stain positively for these proteins. Müller cells with stem cell characteristics also expressed the intermediate filament protein Nestin, a marker of neural stem cells, whilst CE cells did not express this protein. (CE = Ciliary Epithelium, EGF = Epidermal Growth Factor. Scale bars represents 50 μm.)

Comparison between the expression of glial and epithelial cell markers by immortalized Müller glia with stem cell characteristics and non-pigmented CE cells (A) RT-PCR analysis of Müller (MIO-M1 and MIO-M7) and non-pigmented CE cells (CE 6165 and CE 6213) for mRNA expression of retinal pigment epithelial (RPE) markers showed that both cell types expressed mRNA for the RPE cell markers ZO-1, RPE65, MITF-A and MERTK. However, non-pigmented CE cells showed higher expression of these markers than Müller stem cells. mRNA coding for CRALBP, a characteristic marker of Müller glia was expressed by Müller cells but not by CE cells. GAPDH primers were used as control. (B) Immunostaining of Müller and non-pigmented CE cells for markers of RPE. (i) Non-pigmented CE cells showed strong staining with a pan-cytokeratin antibody, whilst no expression of this marker was observed in Müller stem cells. (ii) A small proportion of Müller stem cells stained for RPE65 (less than 10%), whilst all CE cells stained strongly for this protein. (C) Staining for glial markers showed that Müller cells that expressed stem cell properties expressed the proteins CD44 and vimentin, whilst CE cells did not stain for these markers. (CE = Ciliary Epithelium, EGF = Epidermal Growth Factor. Scale bars represents 50 μm.)

Neurogenic ability of non-pigmented CE and Müller stem cells cultured under differentiating conditions for 7 days (A) (i) Morphology of CE and Müller cells cultured under normal culture conditions in the presence of 10% serum. Non-pigmented CE cells were maintained with EGF. (ii) Under adherent conditions in the presence of ECM gel and FGF2, non-pigmented CE cells maintained their epithelial cell morphology, however, a few flattened cells were visible. In contrast, Müller stem cells acquired a neural-like morphology (white arrows). (iii) In the presence of DAPT and FGF2, CE cells ceased to proliferate and did not adopt a neural morphology. However under these conditions a greater proportion of Müller stem cells adopted a neural-like morphology. (Scale bar represents 100 μm.) (B) Western blotting of cell lysates revealed that CE cell preparations cultured under differentiating conditions (CE 6165, CE 6213 and CE 6334) did not express BRN3B, a transcription factor present in post-mitotic retinal ganglion cell precursors. However, increased expression of this protein was observed in Müller stem cells (MIO-M2, MIO-M5 and MIO-M7) cultured under similar conditions. (C) Examination of mRNA for photoreceptor gene expression revealed that in the presence of FGF2 non-pigmented CE cells and Müller stem cells expressed NRL and Rhodopsin, but that only Müller cells expressed S-Opsin. (CE = Ciliary Epithelium, FGF2 = Fibroblast Growth Factor 2.)

Phase-contrast microscopy and proliferation of Müller stem cells and CE cells cultured under adherent conditions in the presence of EGF (A) (i) A small number of phase bright cells isolated from the neural retina were observed attached to the plate one week after isolation, (ii) small colonies of these cells displaying a characteristic glial morphology were observed after 2–3 weeks in culture. (B) (i) Pigmented CE cells showed an aberrant epithelioid morphology after 1 week in culture and did not proliferate. (ii) After 4 weeks in culture, pigmented CE cells were still present but did not proliferate. (C) (i) After 1 week in culture, non-pigmented CE cells had adhered and proliferated. Confluent monolayers exhibited characteristic epithelial morphology at 1 week. (ii) After 4 weeks culture, the rate of proliferation of these cells had decreased and many of them showed aberrant morphology, becoming squamous and showing visible actin filaments (arrows). (D) Non-pigmented CE initially proliferated at a faster rate than those derived from the neural retina. However by passage 6, the majority of cells had died. Cells derived from the neural retina that became spontaneously immortal and later showed stem cell characteristics did not proliferate until after 2–4 weeks in culture, but continued to grow indefinitely in the absence of EGF. (Scale bars represent 100 μm. CE = Ciliary Epithelium, EGF = Epidermal Growth Factor.)