Induction of Functional Photoreceptor Phenotype by Exogenous Crx Expression in Mouse Retinal Stem Cells

Abstract

purpose. This study was undertaken to determine whether exogenous expression of the transcription factor Crx can promote the differentiation of mouse retinal stem cells (RSCs) into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties.

methods. RSCs isolated from mouse ciliary epithelium and maintained in serum-free culture were genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6; POU transcription factor Oct3/4; proliferating cell nuclear antigen [PCNA]); of neuronal markers (nestin, neuron-specific class III β-tubulin [β III Tub] and neurofilament [NF 200]); and of photoreceptor-specific markers (rhodopsin [Rho], cyclic nucleotide-gated cation channel-3 [CNG3], blue-cone opsin, and cGMP phosphodiesterase [PDE]); were evaluated during differentiation by immunocytochemistry and Western blot analysis. Phototransduction cascade activity was assessed by measuring light-induced hydrolysis of cyclic (c)GMP levels with a cGMP enzyme-linked immunoassay.

results. Transient Crx transgene expression was observed in 63% of RSCs. Expression of stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4, was significantly decreased by exogenous Crx expression. Concomitantly, Crx induced expression of the analyzed neuron- and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in RSCs expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels.

conclusions. Crx halted proliferation of RSCs and induced them to differentiate into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. This study demonstrates that Crx can successfully induce RSCs to differentiate into cells with functional photoreceptor phenotypes.

Although it has been known for many years that retinal stem cells (RSCs), with the ability to produce new neurons throughout life, are located at the periphery of the retina in the ciliary marginal zone of fish and amphibians (for review see Refs. ^{1 2} ), it is only recently that their existence in mammals (including humans) has been reported. ^{3 4 5 6 7} The existence of these cells offers hope for the development of therapies for the treatment of human blindness caused by retinal degenerative diseases such as retinitis pigmentosa and macular degeneration. These cells, which persist in a quiescent state beyond embryogenesis throughout life, display the essential properties of stem cells owing to their capacity to self renew and generate multipotent neural or glial cells. However, for retinal stem cell replacement therapy to be developed as an effective and reliable option for the treatment of human retinal degeneration, a greater understanding of the underlying mechanisms implicated in the differentiation of RSCs is necessary. 

In mammals, the ocular ciliary margin, at the peripheral edge of the retina, contains retinal stem cells that are able to proliferate under appropriate conditions. ^{3 4 6 7 8 9 10} These cells are multipotent, and because of their self-renewal capability have been shown to represent a stage antecedent to the retinal progenitors found in the embryonic retina. ¹¹ Given their potential to differentiate along specific retinal lineages when subject to appropriate stimuli, ^{3 4 6 7 9 11 12 13} retinal stem cells from the mammalian ciliary margin are a promising source of cells of therapeutic relevance. Development of protocols that shift phenotypic expression toward the production of specific retinal cell types, especially photoreceptors, is a prerequisite for retinal stem cell replacement to potentially be effective in restoring visual function in blinding diseases. For this purpose, we have addressed the question of how to induce mouse retinal stem cells to become functional photoreceptors. Although extrinsic cues from the environment have recently been shown to regulate retinal stem cell differentiation, ¹⁴ the influence of intrinsic factors is essential to control specific retinal lineage progression in retinal development (for review, see Refs. ^{15 16 17} ). The transcription factor Crx (cone, rod homeobox), is the earliest photoreceptor-specific gene to be expressed and is implicated in the maturation and maintenance of a functional photoreceptor phenotype in vivo. ^{18 19} In particular, Crx controls the expression of photoreceptor-specific genes including phototransduction genes, ²⁰ and mice lacking the gene fail to express proteins implicated in photoreceptor function. ²¹ Furthermore, mutations in human Crx have been associated with congenital blindness and photoreceptor degeneration. ^{22 23 24 25} Significantly for the present study, forced expression of Crx has been shown to induce the generation of photoreceptor-specific phenotypes in stem cells from ciliary- and iris-derived tissues. ^{26 27} To analyze the contribution of this pivotal retinal factor in mediating the retinogenic potential of retinal stem cells, we have therefore tested whether exogenous Crx expression is sufficient for their differentiation into functional photoreceptors displaying light-sensitivity properties. 

Methods

Isolation and Expansion of RSCs

RSCs were isolated from the ciliary epithelium of postnatal day 1 to 5 C57BL/6(J) mice, obtained from Harlan UK (Bicester, UK), and cultured as previously described. ⁴ Briefly, cornea, lens, iris, and neural retina were removed. The ciliary epithelium was dissected, and cell suspensions were prepared using enzymatic digestion at 37°C (trypsin, DNase, collagenase) and mechanical disruption (trituration). The cells were cultured in DMEM/F12, 1× N2 supplement, and 2 mM l-glutamine supplemented with 20 ng/mL EGF (Invitrogen, Carlsbad, CA). 

When required, the differentiation of the RSCs was induced by supplementing the culture medium with 1% fetal calf serum for 8 to 10 days, as previously described. ²⁸  

All animal procedures were performed in compliance with U.K. Home Office regulations and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Assembly of the pMCrx.1 Plasmid

The full-length murine Crx cDNA coding sequence (907 bp) was amplified by PCR, with sequence-specific primers, on total cDNA reverse-transcribed from 1 μg C57BL6/J mouse retinal RNA. The primer sequences were as follows: forward primer (MCRLXV1.1): 5′-CACCATGATGGCATATATGAAC-3′; and reverse primer (MCRLXV1.2): 5′-CAAGATCTGAAACTTCCAGGCA-3′. Thermal profile: 94°C 3 minutes, followed by 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 30 seconds, and a final extension step of 72°C for 5 minutes. The resultant 907-bp product was cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA). The structure of the construct (pMCrx.1) was confirmed by restriction digest analysis and sequencing. 

Electroporation

For each electroporation, 3 to 5 μg DNA/10⁶ RSC suspensions at up to the 11th passage were used. Different electroporation conditions were tested by varying capacitance and voltage (25 μF: 125 V, 250 V; 500 μF: 150 V, 300 V; Gene Pulser II; Bio-Rad, Munich, Germany). After electroporation, the cells were plated in DMEM/F12, 1× N2 supplement, and 2 mM l-glutamine, supplemented with 20 ng/mL EGF (Invitrogen, Carlsbad, CA). 

Efficacy of pMCrx.1 plasmid electroporation was assessed by immunocytochemistry and Western blot. Cells labeled with anti-V5 antibody and counterstained with propidium iodide (PI), were quantified 48 hours after electroporation by counting three random nonoverlapping low-power (×20) images captured with a digital camera (Hamamatsu, Hamamatsu City, Japan) mounted on a microscope (Nikon, Tokyo, Japan). The capture images were obtained at a constant camera setting and exposure time, and the image montages were assembled (Photoshop CS; Adobe, San Jose, CA). Western blot analysis was performed on lysates from cells of the second passage after electroporation (4 weeks after electroporation) and nonelectroporated RSCs at the corresponding passage (the equivalent passage used for electroporation plus two passages). A pcDNA plasmid containing a reporter gene (Invitrogen) was used as a control plasmid. 

Immunocytochemistry

Crx-electroporated RSCs (used at up to second passage after electroporation), and nonelectroporated RSCs at the corresponding passage (equivalent passage used for electroporation plus up to two passages) cultured in differentiation medium, as described previously, ²⁸ were plated on poly-d-lysine-coated slides 24 hours before analysis. The cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed in PBS and incubated with primary antibodies overnight. Antibodies were obtained from (1) Sigma-Aldrich, Ltd. (Dorset, UK): a polyclonal rabbit anti-bovine neurofilament 200 (NF 200) antibody (1:1000), ²⁹ a monoclonal anti-mouse proliferating cell nuclear antigen (PCNA) clone PC10 antibody (1:6000), ³⁰ and a monoclonal anti-mouse rhodopsin (Rho) clone RET-P1 antibody (1:1000) ²⁶ ; (2) Santa Cruz Biotechnology (Santa Cruz, CA): a polyclonal goat anti-mouse cyclic nucleotide-gated (CNG) cation channel 3 (CNG-3) antibody (0.4 μg/mL), ³¹ and a monoclonal anti-human POU transcription factor Oct-3/4 (C10) antibody (1 μg/mL) ³² ; (3) R&D Systems Europe Ltd (Abingdon, UK): a monoclonal anti-rat neuron-specific β-III-tubulin (β III Tub; clone TUJ-1) antibody (1.25 μg/mL) ³³ ; (4) Developmental Studies Hybridoma Bank (DSHB; University of Iowa, Iowa City, IA): a monoclonal anti-rat nestin (clone RAT-401) antibody (1:90) ³⁴ and a monoclonal anti-chicken Pax6 antibody (1:40) ³⁵ ; (5) Invitrogen: a monoclonal anti-V5 epitope (5.5μg/mL) ³⁶ ; and (6) Chemicon (Temecula, CA): a polyclonal rabbit anti-human blue opsin (1.25 μg/mL). ³⁷ A polyclonal rabbit anti-cGMP phosphodiesterase (PDE) antibody (1:500) ³⁸ was a gift from Yee-Kin Ho (University of Illinois, Chicago). The antibodies were visualized as appropriate with goat anti-rabbit IgG, goat anti-mouse IgG, or rabbit anti-goat IgG conjugated to fluorescein (Sigma-Aldrich, Ltd.) at concentrations in accordance with the manufacturer’s recommendations, and the cell nuclei were counterstained with PI. 

Western Blot Analysis

Nonelectroporated RSCs (passage used for electroporation plus two passages) at the corresponding passage of Crx-electroporated RSCs (used up to the second passage after electroporation) were kept in the differentiation medium (as described earlier) before harvesting and were lysed in lysis buffer (50 mM Tris-HCl [pH7.5] 150 mM NaCl, 1% Triton X-100, 2 mM EGTA, 2 mM EDTA, and protease inhibitor cocktail 1 μg/mL; Sigma-Aldrich, Ltd.). Total protein extracts were analyzed by Western blot. ³⁹ Subsequent stripping and detection of actin was used to assess protein loading and transfer. Antibodies were obtained from the same sources as described earlier and were used at the following concentrations: anti-NF 200 antibody (1:2000), ²⁹ anti-PCNA (1:4000), ⁴⁰ anti-rhodopsin (1:2500), ²⁶ anti-CNG-3 (4 μg/mL), ³¹ anti-Oct-3/4 (0.2 μg/mL), ⁴¹ anti-actin (0.2 μg/mL, sc-1615; Santa Cruz Biotechnology), anti-β-TUB (0.25 μg/mL), ⁴² anti-V5 epitope (0.22 μg/mL), ⁴³ anti-blue-cone opsin (2.5μg/mL), ³⁷ and anti-PDE antibody (1:500). ⁴⁴ In addition, a polyclonal rabbit anti-Pax-6 antibody (1:1000) was a gift from Peter Gruss (University of Göttingen, Germany). ⁴⁵ The immunoreactive proteins were visualized with horseradish-peroxidase–linked donkey anti-goat antibody, goat anti-mouse or goat anti-rabbit antibody, and enhanced chemiluminescence detection (Santa Cruz Biotechnology). 

Functional Characterization of Crx-Transduced RSCs

Functional phototransduction pathways were assessed by measuring the level of cGMP hydrolysis with an enzyme immunoassay kit, according to the manufacturer’s protocol (Biotrack [EIA] System; GE Healthcare, Buckinghamshire, UK) in RSCs non-electroporated and electroporated at up to the second passage of culture in differentiation medium for 24 hours (see above), kept in the dark, or maintained in ambient daylight. When used, the PDE inhibitor, IBMX (3-isobutyl-1-methylxanthine) (Sigma-Aldrich, Ltd.), was added (50 mM) 48 hours before determination of cGMP levels. 

Results

Crx Gene Transfer into RSCs

To determine the effect of exogenous Crx expression on mouse retinal stem cells, we first constructed a plasmid (pMCrx.1) that contained the full-length murine Crx cDNA coding sequence (907 bp) driven by the CMV IE promoter and upstream of the V5-His6 Tag (Fig. 1A) . 

In preliminary experiments, the use of cationic lipids (lipofectamine) resulted in very low level of RSCs transfection (<2%) and a high rate of cell death (data not shown). As an alternative method, electroporation was considered for delivering pMCrx.1 into the RSCs. Optimal electroporation conditions were defined by varying capacitance and voltage (25 μF: 125 V, 250 V; 500 μF: 150 V, 300 V; Figs. 1B 1C 1D 1E 1F ). Expression of exogenous Crx was monitored after electroporation by using immunocytochemistry to detect V5-tagged protein. The percentage of cells expressing the V5 tag was determined 48 hours after electroporation (Figs. 1B 1C 1D 1E 1F) . No nonspecific immunofluorescence was observed in cells without primary antibody in this or any other of the immunocytochemical analyses described below (data not shown). No V5-immunoreactivity was detected in RSCs not electroporated or electroporated with a control plasmid (data not shown). A voltage of 125 V and a capacitance of 25 μF were found to give more than 60% (62.9% ± 5.5%, n = 3, P < 0.003) of V5-positive cells (Fig. 1F) . More than 50% (53.6% ± 3.9%, n = 3, P < 0.002) of cells were also found to be positive at 150 V and 500 μF and no statistically significant difference between the two conditions was observed. However, the immunolabeling was more intense in the first conditions (Fig. 1Bcompared with 1D). Increasing the voltage reduced the transfection efficiency by ∼40% at 25 μF capacitance (from 62.9% at 125 V to 20% at 250 V; statistically significant difference between 125 and 250 V, P < 0.002, unpaired t-test) and by ∼30% at 500 μF capacitance (from 53% at 150 V to 23% at 300 V; statistically significant difference between 150 and 300 V, P < 0.003, unpaired t-test). To ascertain whether exogenous Crx expression was sustained in electroporated RSCs maintained continuously in culture, V5 tag expression was analyzed by Western blots (Fig. 1G) . In cell lysates obtained from second-passage cultures (4 weeks after electroporation), V5 expression was immunodetectable in protein extracts of cells electroporated in all four conditions tested (Fig. 1G) . No expression of V5 was detected in RSCs maintained in identical culture conditions, which were either nonelectroporated (data not shown) or electroporated with a control plasmid (Fig. 1G) . Cells at up to the second passage after electroporation at 125 V and 25 μF were used in the following experiments. 

Effect of Exogenous Crx Gene Expression on Markers of RSC Proliferation and Differentiation

The effects of Crx on the expression of cell cycle–related proteins and markers of stem cell status were examined by immunocytochemistry (Fig. 2)and Western blot (Fig. 3) . Comparisons of nonelectroporated and control plasmid–electroporated RSCs (pCDNA plasmid containing the LacZ reporter gene) showed no statistically significant difference in the expression levels, or observable differences in the immunocytologic patterns, of these markers by Western blot analysis (Figs. 3A 3B 3C)or immunocytochemistry, respectively (data not shown). Immunoreactivity for the homeodomain transcription factor Pax6, which is expressed developmentally in embryonic retina, was only very faintly detectable in Crx-electroporated cells (Fig. 2B)compared with moderate levels detected in control nonelectroporated RSCs (Fig. 2A) . This result was confirmed by Western blot analysis. Several Pax6 isoforms were detected as previously reported. ^{46 47 48} In particular, the Pax6 p46 isoform (molecular mass: 46 kDa) levels were found to be similar in nonelectroporated RSCs and cells electroporated with a control plasmid, but a lower level of expression (40% of RSCs expression level; P < 0.02, unpaired t-test) was observed in Crx-electroporated cells (Fig. 3A) . Similarly, immunodetection of proliferating cell nuclear antigen (PCNA), a cell-cycle protein present in dividing cells, showed a decrease in expression after electroporation (Figs. 2C 2D) . Western blot analysis confirmed that a lower level of expression (38% of RSCs expression level; P < 0.002, unpaired t-test) was detected in Crx-electroporated cells. Although PCNA expression decreased slightly in control plasmid–electroporated RSCs compared with nonelectroporated cells, this difference was found not to be statistically significant (Fig. 3B ; P > 0.05). Expression of the POU transcription factor Oct3/4, a master regulator of cell pluripotency, was also decreased in Crx-electroporated cells by immunolabeling (Figs. 2E 2F) . This result was supported by Western blot analysis, Oct3/4 expression decreased by 31% in Crx-electroporated cells compared with RSCs (Fig. 3C ; P < 0.03, unpaired t-test). 

Effect of Exogenous Crx Gene Expression on Neuronal Markers in RSCs

The effect of Crx on the expression of markers of early and late neuronal differentiation was examined by immunocytochemistry (Fig. 4)and Western blot analysis (Fig. 3) . Comparisons of nonelectroporated and control plasmid–electroporated RSCs showed no statistically significant difference in the expression levels, or observable differences in the immunocytologic patterns, of these markers by Western blot analysis (Figs. 3D 3E)or immunocytochemistry, respectively (data not shown). The neuronal stem cell marker protein nestin was expressed in both control and transfected cells, although fewer cells were labeled after Crx transfection (Fig. 4B). In contrast, increased immunoreactivity to class III β-tubulin (β III Tub) and neurofilament (NF 200), both late neuronal markers, was observed in Crx-transfected cells (Figs. 4D 4F) . Western blot analysis confirmed these results, the levels of β III Tub and NF 200 expression were significantly higher in Crx-electroporated cells than in nonelectroporated and plasmid control–electroporated RSCs (respectively, 2.9 times higher, P < 0.05; and 3.5 times higher, P < 0.03; unpaired t tests; Figs. 3D 3E ). 

Effect of Exogenous Crx Gene Expression on Photoreceptor-Specific Markers in RSCs

To determine whether Crx promotes differentiation into the photoreceptor phenotype, expression of photoreceptor-specific genes was assessed in Crx-electroporated RSCs by immunocytochemistry (Fig. 5)and Western blot analysis (Fig. 3) . Comparisons of nonelectroporated and control plasmid–electroporated RSCs showed no statistically significant difference in the expression levels or observable differences in the immunocytologic patterns, of these markers by Western blot analysis (Figs. 3F 3G 3H 3I)or immunocytochemistry, respectively (data not shown). Immunolabeling of rhodopsin (Rho), cGMP phosphodiesterase (PDE), blue-cone opsin, and cyclic nucleotide-gated cation channel-3 (CNG3) was increased in Crx-electroporated cells (Figs. 5B 5D 5F 5H) . In addition, double immunostaining with anti-V5 tagged protein and either anti-blue cone opsin or anti-PDE confirmed coexpression of the Crx transgene with photoreceptor-specific markers (Fig. 6) . The expression of these markers was quantified by Western blot analysis (Figs. 3F 3G 3H 3I) . The expression of the four photoreceptor-specific markers was significantly higher in Crx-electroporated cells than in non-electroporated and plasmid control–electroporated RSCs (Rho: 3.3 times, P < 0.01; PDE: 2.0 times, P < 0.04; blue opsin: 3.0 times, P < 0.04; CNG3: 3.3 times, P < 0.03; unpaired t-tests). 

Effect of Exogenous Crx Gene Expression on Differentiation of RSCs into Photoreceptor-Specific Functional Phenotype

To establish whether Crx-electroporated RSCs exhibit a phototransduction function, we evaluated the physiological characteristics of these cells on differentiation. In vivo, visual transduction is mediated by complex biochemical pathways that precisely regulate cGMP levels in photoreceptors. In an in vitro system, the sensitivity of cGMP levels to light can be taken as a measure of an activatable phototransduction cascade. We assessed the effect of exogenous Crx expression on cGMP levels by immunoassay in cell extracts obtained from cells subjected to ambient light or maintained in the dark (Fig. 7) . No difference in cGMP levels, between light and dark conditions, was observed in nonelectroporated and plasmid control–electroporated RSCs (Fig. 7) . However, an increase in cGMP level was observed when Crx-electroporated RSCs were kept in the dark (3.2 times higher, P < 0.03; unpaired t tests; Fig. 7A ). cGMP is hydrolyzed by a family of PDEs. To evaluate the total amount of cGMP hydrolyzed by the various PDEs present in the RSCs, a nonspecific inhibitor (IBMX) was added. ⁴⁹ Increased cGMP levels (6.0 times higher) were observed in nonelectroporated and plasmid control–electroporated RSCs in light and dark conditions, and in the light-exposed Crx-electroporated cells (Fig. 7B) . The cGMP level difference observed between Crx-transfected RSCs in light and dark conditions was abolished by addition of the PDE inhibitor (Fig. 7) . The amount of cGMP hydrolyzed by light-sensitive PDE corresponds to the difference between the levels in light and dark conditions (∼30 fmol/10⁶ cells) in Crx electroporated cells (Fig. 7) , whereas the total cGMP hydrolyzed by all PDEs corresponds to ∼65 fmol/10⁶ cells (the difference between the cGMP levels in the presence and absence of IBMX in the light). Therefore, light-sensitive PDE represents ∼46% (30/65) of the total PDE activity in Crx electroporated RSCs (Figs. 7A 7B) . 

Discussion

In the present study, we have demonstrated that murine retinal stem cells can be induced by exogenous expression of Crx to differentiate into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. 

Genetic modification of the RSCs was achieved by electroporation, a method shown to be more effective than liposome-based methods for transferring genes in embryonic stem cells (Ref. ⁵⁰ and the present study). Our results indicated that efficient transient transgene expression can be achieved by electroporating RSCs. Expression of exogenous Crx by 63% of the RSCs was found to be sufficient to enable detection of significant changes in the differentiation and maturation processes of these cells. Of importance, expression of the key stem cell markers of proliferation and pluripotency, Pax6 and Oct3/4, was found to be unaltered in RSCs electroporated with a control plasmid, suggesting that these properties were not affected by the procedure. Although PCNA expression appeared to decrease slightly in cells electroporated with a control plasmid compared with RSCs not electroporated, this difference was not statistically significant, indicating that electroporation does not alter significantly their proliferative properties. However, transient expression of Crx was found to decrease significantly the level of expression of these same stem cell markers. Pax6, a paired domain and homeodomain-containing transcription factor, controls eye development at an early stage and is essential for the formation of nearly all retinal cell types except amacrine cells (for review, see Ref. ⁵¹ ). Since Pax6 is necessary for RSC proliferation and expansion, ^{52 53 54} the downregulation observed when exogenous Crx is expressed suggests that the cells have stopped proliferating. Adult retinal stem cells of the retina’s marginal zone that continue to proliferate into adulthood, have been shown to coexpress Pax6 and PCNA. ⁵ Therefore, concomitant downregulation of PCNA and Pax6 expression in RSCs supports the hypothesis that exogenous Crx expression decreases proliferation. Expression of the POU homeodomain transcription factor Oct-3/4, a key regulator of self-renewal and differentiation in embryonic stem cells, ^{54 55 56 57} is also downregulated in Crx-expressing RSCs. Although doubt has been cast ⁵⁸ on the role of Oct3/4 as a pure stem cell marker in adult stem cells, ^{32 59 60} its decreased expression, taken in conjunction with downregulation of Pax6 and PCNA, strongly suggests that exogenous Crx expression impacts negatively on the RSCs’ pluripotency and proliferation properties. 

Furthermore, downregulation of these genes coincides with the upregulated expression of proteins specific for neuronal lineages (class III β-tubulin, NF 200), suggesting that exogenous-Crx expression induces neuronal differentiation. Because Crx has been shown to control expression of a core set of photoreceptor genes, ²⁰ expression of photoreceptor-specific markers (cone and rod: PDE, rod-specific: rhodopsin, cone-specific: blue opsin, CNG3) was assessed to characterize further the neuronal phenotype of these Crx-transduced RSCs. Expression of these genes was found to be increased, suggesting that exogenous expression of Crx induces RSCs to differentiate into photoreceptor phenotypes. Colocalization of exogenous Crx with either blue cone opsin or PDE supports this hypothesis. The existence of cells expressing specific photoreceptor markers but apparently lacking exogenous Crx expression implies one or more of three possible scenarios: (1) The expression of the Crx transgene has undergone downregulation after induction of differentiation into photoreceptor phenotypes; (2) transgenic Crx expression results in an extrinsic, paracrine-like effect that promotes differentiation of untransduced cells; (3) and/or the differentiation medium can act on a proportion of cells to induce expression of the detected photoreceptor markers (Ref. ²⁸ and the present study). Isolation of RSC clones stably expressing the transgene will help us to establish more precisely the effect of exogenous Crx expression in these RSCs. Nonetheless, upregulation of rod and blue cone opsin protein expression suggests that both rod and cone phenotypes are induced. These proteins were found to show cytoplasmic localization in the RSCs, indicating that contrary to photoreceptors in the mature retina, these cells lack polarity. Akagi et al. ²⁷ have reported a similar rod opsin immunodistribution in iris-derived cells transfected with Crx. Diffuse localization of cone opsins has also been detected in the mouse cone photoreceptor cell line 661W, suggesting that photoreceptors lose their polarity when cultured in vitro. ⁶¹ In addition, that retinal pigment epithelium (RPE) transplantation induces regeneration of photoreceptor outer segments in RCS rats ⁶² indicates that physical contact with RPE is needed for photoreceptor polarity. Whether the RSCs expressing exogenous Crx acquire this property when they integrate into mature retina, as shown for iris-derived cells expressing Crx, ²⁷ remains to be ascertained. Nevertheless, a significant increase in light-induced cGMP hydrolysis was observed in Crx-transduced RSCs, suggesting that the phototransduction cascade can be activated by light stimulus. In the retina, light absorbed by the photoreceptors triggers a cascade of reactions that initiate cGMP hydrolysis by PDE. ^{63 64 65 66} PDE inhibitors (including IBMX) induce inhibition of the light responsiveness of photoreceptors consistent with elevated levels of cGMP. ⁴⁹ IBMX PDE inhibitor targets not only photoreceptor-specific PDE but all members of the PDE family. ⁴⁹ Increased levels of cGMP detected in IBMX-treated Crx-transduced RSCs in the dark relative to nontreated cells most likely reflect activity of other, nonphotoreceptor PDEs. Light-sensitive PDE activity represents ∼46% of the total PDE activity in the Crx-transduced RSCs. Total levels of cGMP are equal in all the cell lines in the presence of IBMX. Therefore, in Crx-electroporated RSCs, activity of non–light-sensitive PDEs has decreased correspondingly by ∼46% of the total PDE activity. These data suggest that Crx has induced RSCs to differentiate into cells that have not only lost 46% of their original non–light-sensitive PDE activities, but most important have acquired light-sensitive PDE activity. Taken together with the evidence of upregulated expression of key components of the visual transduction cascade, these observations strongly support the hypothesis that exogenous Crx induces RSCs to differentiate into functional photoreceptor phenotypes. 

This study lends support to the notion that genetic modification of RSCs can be used efficiently for the production of specific retinal cell types. In the future, RSC clones stably expressing Crx will enable us to evaluate their competence to integrate, survive, and display physiological photoreceptor function in the retina in vivo. 

 Supported by the British Retinitis Pigmentosa Society and the Iris Fund for Prevention of Blindness.

 Submitted for publication July 2, 2007; revised September 11, 2007; accepted November 21, 2007.

 Disclosure: C. Jomary, None; S.E. Jones, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Optimization of exogenous Crx expression after electroporation with the pMCrx.1 plasmid. (A) Representation of part of the plasmid clone pMCrx.1. A PCR-amplified full-length murine Crx coding sequence cDNA (907 bp) was inserted into the pCDNA 3.1D/V5-His-TOPO vector, downstream of the CMV IE promoter and upstream of the V5-His 6Tag. A variety of electroporation parameters was tested: (B) 125 V, 25 μF; (C) 250 V, 25 μF; (D) 250 V, 500 μF; and (E) 300 V, 500 μF. Cell nuclei were counterstained with PI. Exogenous Crx expression was measured by detecting V5-tagged protein 48 hours after electroporation and was quantified by counting three random nonoverlapping images (F). Results are expressed as the mean ± SEM (n = 3). Statistically significant differences: *P < 0.003, •P < 0.002; unpaired t-test. Exogenous Crx expression was analyzed at 4 weeks after electroporation by Western blots of cell lysates from RSCs electroporated with pMCrx.1 or control (Cont) plasmid. Densitometric analysis of the ratio of V5 to actin is shown (G). Scale bar, 100 μm.

Figure 2.

 

Effect of exogenous Crx gene expression on the expression of proliferation and differentiation markers in RSCs. Immunocytochemical labeling of the homeodomain transcription factor Pax6 (A, B), of the proliferating nuclear antigen (PCNA) (C, D), and the POU transcription factor Oct3/4 (E, F) in RSCs (A, C, E) and Crx-electroporated RSCs (B, D, F). Cell nuclei were counterstained with PI (C–F). Scale bar, 100 μm.

Figure 3.

 

Western blot analysis of stem cell markers (A–C), neuronal markers (D, E), and photoreceptor markers (F–I) in nonelectroporated (RSC), Crx-electroporated (CRX), and control plasmid–electroporated (Cont) RSCs. Protein loading was assessed with actin (J). Densitometric analysis of the ratio to actin of (A) Pax6 isoform p46 (*P < 0.02, unpaired t-test); (B) PCNA (*P < 0.002, unpaired t-test); (C) Oct3/4 (*P < 0.03, unpaired t-test); (D) neuron-specific class β III Tub (*P < 0.05, unpaired t-test); (E) NF 200 (*P < 0.03, unpaired t-test); (F) Rho (*P < 0.01, unpaired t-test); (G) cGMP PDE (*P < 0.04, unpaired t-test); (H) blue cone opsin (P < 0.04, unpaired t-test); and (I) CNG3 (*P < 0.03, unpaired t-test); Results are expressed as the mean ± SEM (n = 3).

Figure 4.

 

Effect of exogenous Crx gene expression on neuronal markers expression in RSCs. Immunolabeling of nestin (A, B), class III β-Tub (C, D), and NF 200 (E, F) in RSCs (A, C, E) and Crx-electroporated RSCs (B, D, F). Cell nuclei were counterstained with PI (A–F). Scale bar, 100 μm.

Figure 5.

 

Effects of exogenous Crx gene expression on photoreceptor-specific markers in RSCs. Immunolabeling of Rho (A, B), cGMP PDE (C, D), blue cone opsin (E, F), and CNG3 (G, H), in RSCs (A, C, E, G) and Crx-electroporated RSCs (B, D, F, H). Cell nuclei were counterstained with PI. Scale bar, 100 μm.

Figure 6.

 

Expression of photoreceptor-specific markers by RSCs expressing exogenous Crx gene. RSCs electroporated with pMCrx.1 expressed blue cone opsin (A, B) and cGMP PDE (C, D). Double immunolabeling in RSCs showed V5-positive cells expressing exogenous Crx (A, C), blue cone opsin (B), and cGMP PDE (D). Arrows: RSCs double-labeled with anti-V5 and anti-blue cone opsin (A, B), or anti-V5 and anti-PDE (C, D). Arrowheads: cells that were not double labeled (C, D). Scale bar, 100 μm.

Figure 7.

 

Effect of exogenous Crx gene expression on cGMP levels in nonelectroporated (RSC), Crx-electroporated (CRX), and control plasmid–electroporated (Cont) RSCs. Cells were exposed to light or kept in the dark in control conditions (A) and in the presence of an inhibitor of phosphodiesterases (IBMX) (B). Results are expressed as the mean ± SEM (n= 3). *P < 0.03 unpaired t-test.

The authors thank Jason Cullen for technical assistance.