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Stem Cells 2005;23:424-432 www.StemCells.com
© 2005 AlphaMed Press

Effects of Ciliary Neurotrophic Factor on Differentiation of Late Retinal Progenitor Cells

Tasneem Zahira, Henry Klassenb, Michael J. Younga

a Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, USA, and
b Children’s Hospital of Orange County, Orange, California, USA

Key Words. Retinal progenitor cells • Ciliary neurotrophic factor • Bipolar • Glial • Differentiation

Correspondence: Michael J. Young, Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, Massachusetts 02114, USA. Telephone: 617–912–7419; fax: 617–912–0101; e-mail: mikey{at}vision.eri.harvard.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ciliary neurotrophic factor (CNTF) has been shown to be a potent regulator of retinal cell differentiation. The present study was undertaken to investigate the effects of CNTF on in vitro differentiation of expanded late retinal progenitor cells. Retinal progenitor cells used in these studies were isolated from the neural retina of postnatal day-1 green fluorescent protein (GFP) transgenic mice. The resulting GFP-positive neurospheres were dissociated into a single-cell suspension and grown on poly-D-lysine/laminin-coated tissue culture flasks or slides to generate adherent retinal progenitor cells. These adherent cells were treated with 20 ng/ml of CNTF for up to 14 days, and expression of specific retinal cell markers was determined by immunocytochemistry, reverse transcription–polymerase chain reaction (RT-PCR), and immunoblot analysis. In vitro studies showed that CNTF treatment of late retinal progenitor cells resulted in changes in cellular morphology. Immunocytochemical studies showed an increase in the proportion of cells expressing markers of bipolar cells but not rod differentiation. In addition, an increase in the proportion of cells expressing glial cell markers was observed. RT-PCR analysis showed downregulation in Hes1, Nestin, Notch1, and Pax6 transcripts along with a concomitant increase in protein kinase C (PKC){alpha} and glial fibrillary acidic protein (GFAP) transcripts. These findings were confirmed by immunoblot analysis, where downregulation in Nestin expression and simultaneous upregulation in PKC{alpha} and GFAP were observed. The data indicate that CNTF treatment of multipotential late retinal progenitors increases the proportion of cells that express markers of bipolar neurons and glia.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The vertebrate retina is composed of seven distinct cell types that include ganglion cells, amacrine cells, bipolar cells, horizontal cell, rod and cone photoreceptor cells, and Müller glial cells. Thymidine birth-dating studies have shown that each cell type emerges from retinal progenitor cells in an invariant temporal sequence, with some overlap in the generation of certain cell types [1]. Retinal ganglion cells, cones, horizontal cells, and amacrine cells are born during early retinogenesis, whereas the bipolar cells, rods, and Müller glial cells are born during late retinogenesis. Lineage tracing studies have shown that progenitor cells in the developing retina, which give rise to the various cell types, are multipotential, and their choice of cell fate is governed not only by intrinsic but also extrinsic signals from the microenvironment [2, 3].

To further investigate the influence of extrinsic factors on the differentiation of retinal progenitor cells, in vitro studies have been carried out in which several stimulatory molecules, including basic fibroblast growth factor [4], glial cell line–derived growth factor [4, 5], retinoic acid [6, 7], taurine [8], and sonic hedgehog [9], have been identified as factors that influence the generation of rod photoreceptors. In addition to these factors, ciliary neurotrophic factor (CNTF) has been shown to be involved in rod photoreceptor differentiation. CNTF is a pleiotropic growth factor with various potential functions in the developing retina. It has been shown to promote the survival of embryonic chick ciliary ganglion neurons [10], to inhibit the proliferation of sympathetic precursor cells and induce a change from adrenergic to cholinergic neurons [11], and to promote differentiation of glial progenitor cells into astrocytes and oligodendrocytes [12, 13]. It belongs to a family of cytokines that include interleukin-6 (IL-6), IL-11, leukemia inhibitory factor, and oncostatin M. The effects of CNTF are mediated by a tripartite receptor complex consisting of two signal transducing subunits (leukemia inhibitory receptor ß and gp130), which are also components of other cytokine receptors, and the CNTF-specific ligand-binding {alpha} [14].

CNTF has been shown to regulate the differentiation of rod photoreceptors during a transient period of development in the vertebrate retina; however, CNTF has opposite effects on rod photoreceptor differentiation in the chick and rat [15, 16]. In the avian retina, CNTF promotes the maturation of early, postmitotic photoreceptors into rod photoreceptors that express rhodopsin, whereas in the rodent retina, CNTF acts as a negative regulator of rod photoreceptor differentiation in vitro [15–17]. In these studies, the CNTF-induced decrease in rhodopsin expression in rat retinal explant cultures was accompanied by an increase in the expression of bipolar cell markers (PKC{alpha} and mGluR6) in rod precursors located in the photoreceptor layer [18]. Also, there was a small increase in cells expressing the glial cell marker glial fibrillary acidic protein (GFAP). This may be due to increased survival or proliferation of retinal astrocytes but could also be explained by the upregulation of GFAP expression by Müller glial cells [18].

The present study was undertaken to investigate the effects of CNTF on in vitro differentiation of late retinal progenitor cells. Because the progeny of late retinogenesis includes rod photoreceptors, bipolar neurons, and Müller glial cells, we sought to determine whether CNTF treatment of late retinal progenitor cells selectively induces bipolar differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and Culture of Late Retinal Progenitor Cells
Retinal progenitor cells (RPCs) were isolated from the neural retina of postnatal day-1 GFP transgenic mice [19]. Briefly, retinas were harvested from newborn GFP transgenic mice [20] and subjected to several cycles of collagenase digestion to dissociate the tissue. Cells were then forced through a nylon mesh, centrifuged, and resuspended in complete medium containing Dulbecco’s modified Eagle’s medium/Ham’s F12 1:1 (Omega Scientific) supplemented with 100 µg/ml N2 neural supplement (Gibco), 2 mM L-glutamine (Sigma), 2,000 U nystatin (Gibco), 100 µg/ml penicillin-streptomycin (Sigma), and 20 ng/ml epidermal growth factor (recombinant human epidermal growth factor [EGF]; Gibco). GFP+ neurospheres formed within the first 3 days and were passaged at regular intervals. To assess the capacity for self-renewal, spheres were broken up and plated as single cells. Individual cells formed new spheres over a period of 2 to 5 days. Spheres labeled positively for Nestin (a marker for neural progenitor cells) and Ki67 (a marker for cell proliferation) [19].

Differentiation of Late Retinal Progenitor Cells
The GFP+ neurospheres were dissociated into single-cell suspension and grown on poly-D-lysine and laminin-coated tissue culture plates to generate adherent retinal progenitor cells that were subsequently differentiated. Briefly, passage-25 cells from confluent T75 flasks were trypsinized and resuspended into single-cell suspension (200,000 cells/ml). These cells were then seeded onto Biocoat poly-D-lysine/mouse laminin-coated eight-well culture slides or flasks (Becton Dickinson Labware) and allowed to grow for 18 to 24 hours at 37°C in complete medium. The complete cell culture medium containing EGF was then removed from the cells, and the cells were washed with Ca2+ and Mg2+ free Hanks’ balanced salt solution (Gibco). Fresh medium containing CNTF (20 ng/ml; R&D Systems) but no EGF was added to the experimental wells/flasks, whereas the controls were treated with medium containing no CNTF. The medium was changed every 3 to 4 days for up to 14 days. The cells grown in flasks were used for reverse transcription–polymerase chain reaction (RT-PCR) and immunoblot analysis, whereas the cells grown on culture slides were fixed at 14 days after CNTF treatment for immunocytochemical studies.

Immunocytochemistry
RPCs cultured on Biocoat poly-D-lysine/mouse laminin culture slides were processed according to standard protocols. Briefly, cells were fixed in 4% paraformaldehyde (pH 7.2) in 0.1 M phosphate-buffered saline (PBS) and blocked in PBS containing 2% bovine serum albumin (BSA) and 0.5% Triton X-100. They were then incubated with appropriate primary antibodies (Table 1Go) for 2 hours at room temperature. Antibody incubations were conducted in PBS containing 2% BSA. Cells were washed three times for 10 minutes each with PBS and incubated with species-specific secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch Laboratory, Inc.) for 1 hour at room temperature. Subsequently, cells were washed three times with PBS, coverslipped in 2.5% polyvinyl alcohol/DABCO (1,4 diazabicyclo{2,2,2} octane), and examined in an epifluorescence microscope (Nikon Eclipse, E800).


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  		Table 1. Primary antibodies used for immunocytochemistry
 
Quantitative immunocytochemistry was performed by counting a total cell number of at least 200 cells per well in randomly selected fields using 4',6-Diamidino-2-phe-nylindole labeling. Each determination was performed in quadruplicate (n = 4), and each experiment was repeated at least three times. Note that the number of Nestin+ RPCs in the CNTF-treated cultures may have been underestimated because of downregulation of protein expression resulting in reduced intensity of staining.

RT-PCR Analysis
Total RNA was isolated from RPCs (106 cells per sample) grown in the absence or presence of CNTF with RNAqueous-4PCR kit (Ambion). RT reactions were set up using the RETROscript kit (Ambion). A two-step RT-PCR approach, involving an initial step of heat denaturation of RNA to obtain cDNA followed by a step of PCR amplification using gene specific primers (Table 2Go), was used. The thermocycler program that was used for PCR amplification included a hot start (95°C for 1 minute) followed by denaturation at 94°C for 30 seconds, annealing at specific temperatures (Table 2Go) for 30 seconds for 30 cycles, and extension at 72°C for 30 seconds for 30 cycles, followed by a final extension at 72°C for 5 minutes. RT-PCR reactions (cDNA) were run on 2% agarose gels (containing ethidium bromide at a final concentration of 0.5 µg/ml) against a 100-bp ladder, and the products were visualized under UV light. The amount of cDNA was normalized based on the signal from constitutively expressed S15 mRNA, which encodes a small ribosomal subunit protein, to analyze relative expression of different mRNAs.


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  		Table 2. Primers used for reverse transcription–polymerase chain reaction analysis
 
Immunoblot Analysis
RPCs, grown in the absence or presence of CNTF, and B6 mice retinas (4 weeks) were homogenized in lysis buffer (1% Triton-X 100, 10 mM Tris-HCl [pH 7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF) containing protease inhibitor cocktail (1:100 dilution; Sigma) and phosphotase inhibitor (1:100 dilution; Sigma). Protein levels of total cell lysates were quantified with a protein assay kit (Bio-Rad). The protein samples (20 µg) were separated on poly-acrylamide gels (NuPage; Invitrogen) for 40 minutes at 160 V and transferred to polyvinylidene difluoride membranes (Invitrolon; Invitrogen) for 60 minutes at 30 V. After transfer, the membranes were blocked in 5% nonfat dry milk in tris-buffered saline-Tween-20 (10 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 30 minutes. The blots were incubated with the following primary antibodies: GFAP (1:1,000 dilution, Chemicon), Nestin (1:20 dilution, DSHB), PKC{alpha} (1:200 dilution; Santa Cruz Biotechnology), and recoverin (1:1,000 dilution; Chemicon). Subsequently, blots were incubated with horseradish peroxidase–conjugated species-specific secondary antibodies, and the signals were visualized with the ECL Western blotting detection system (Amersham Biosciences).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Retinal Progenitor Markers by Late RPCs
The GFP+ neurospheres (Fig. 1AGo) were dissociated into single-cell suspension and grown on poly-D-lysine and laminin-coated tissue culture plates to generate adherent retinal progenitor cells (Fig. 1BGo). The proliferative potential and expression of neural progenitor cell markers was confirmed by immunostaining with Ki67 (a marker for cell proliferation) and Nestin (a marker for neural progenitor cells). Ki67 staining, like bromodeoxyurindine pulse labeling, is used to determine the proportion of cells that are in the S phase of the mitotic cycle. Because only a subset of cells from the total cell population enter the cell cycle at any given time, it is unlikely that > 45% of cells stain positive for Ki67, even in case of a highly proliferative cell line. In the present study, 42.2% of the adherent RPCs expressed Ki67 (Table 3Go; Fig. 2AGo), whereas > 86.5% expressed Nestin (Table 3Go; Fig. 2BGo), confirming that these cells shared the progenitor cell characteristics (expression of proliferative and progenitor cell markers) of the GFP+ neurospheres from which they were derived. The expression of Ki67 (Fig. 2CGo) and Nestin (Fig. 2DGo) was downregulated by adherent RPCs upon differentiation.



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  		Figure 1. Late RPCs grown in vitro as neurospheres (A) and adherent cells (B). RPCs were maintained as neurospheres when cultured in complete media. (A): Fluorescent images of GFP+ spheres (x 200). These neurospheres were dissociated into single-cell suspension and grown on poly-D lysine/laminin-coated tissue culture slides to generate adherent RPCs. (B): Fluorescent images of GFP+ adherent RPCs (x 200). Abbreviations: GFP, green fluorescent protein; RPC, retinal progenitor cell.

 

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  		Table 3. Quantitative evaluation of effect of CNTF on the expression of different cell markers
 


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  		Figure 2. Expression of cell proliferation and neuronal progenitor markers by late RPCs grown as adherent cells before (A, B) and after (C, D) CNTF treatment. (A, B): Fluorescent images of adherent RPCs illustrating immunoreactivity for Ki67 (A) (x 200) and Nestin (B) (x 200) in the absence of CNTF. (C, D): Downregulation in Ki67 (C) (x 200) and Nestin (D) (x 200) expression by RPCs as a result of CNTF treatment. The number of cells expressing these markers decreased dramatically in the presence of CNTF. Abbreviations: CNTF, Ciliary neurotrophic factor; RPC, retinal progenitor cell.

 
CNTF Induced Changes in the Morphology and Rate of Proliferation of Late RPCs
RPC neurospheres were dissociated and plated onto a substrate to generate adherent cells. The adherent RPCs continued to proliferate and formed a monolayer. Most of these cells remained morphologically undifferentiated, whereas a few gave out short processes (Fig. 3AGo). After CNTF treatment, the proliferation of adherent RPCs decreased dramatically. The percentage of RPCs expressing Ki67 decreased from 42.2% before treatment to less than 2.9% after treatment with CNTF (Table 3Go). Many of these cells adopted a bipolar morphology and extended long thin neurite-like processes, forming a network between neighboring cells (Fig. 3BGo).



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  		Figure 3. CNTF induced changes in the morphology of late RPCs. (A, B): Phase-contrast images of adherent RPCs before and after CNTF treatment. Before CNTF treatment, RPCs grew as an undifferentiated monolayer, with some cells giving out short processes (A) (x 200). However, after CNTF treatment, most of the cells adopted a bipolar morphology, with long neurite-like processes that formed a network between cells (B) (x 200). Inset shows cells depicting bipolar morphology at higher magnification from the same culture. Abbreviations: CNTF, Ciliary neurotrophic factor; RPC, retinal progenitor cell.

 
Quantitative Evaluation of Effect of CNTF on Bipolar, Glial, and Progenitor Marker Expression Using Immunocytochemistry
The effect of CNTF on PKC{alpha} and GFAP expression was determined by immunocytochemistry (Table 3Go; Fig. 4Go). After 14 days of CNTF treatment, the percentage of RPCs expressing PKC{alpha} increased from 12.8% to 41.1% (Table 3Go). Most of the PKC{alpha}-positive cells in the CNTF-treated cultures exhibited bipolar morphology (Figs. 4J–4LGo), whereas those in the untreated cultures remained undifferentiated (Figs. 4G–4IGo). CNTF also had an inductive effect on GFAP expression. The percentage of cells expressing GFAP increased significantly, from 8.2% to 48.3% (Table 3Go). The GFAP-positive cells in RPC cultures grown in the absence of CNTF appeared undifferentiated (Figs. 4A–4CGo), whereas those grown in the presence of CNTF displayed various morphologies, including cells extending many thin, neurite-like processes (Figs. 4D–4FGo). In contrast to the increase observed in the percentage of cells expressing bipolar and glial markers, CNTF treatment decreased the percentage of Nestin-positive cells from 86.5% to 48.5% (Table 3Go). In addition, RPCs grown in the absence of CNTF (Fig. 2BGo) displayed more intense Nestin staining compared with those grown in the presence of CNTF (Fig. 2DGo).



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  		Figure 4. Effect of CNTF on late RPC expression of neuronal and glial markers. RPCs grown in the absence (A–C, G–I) (x 600) or presence (D–F, J–L) (x 600) of CNTF were examined for GFAP and PKC{alpha} expression. (A, D): Fluorescent images of GFAP immunoreactivity; (B, E): GFP expression in the same RPCs. (C, F): Combined images of (A, B) and (D, E). CNTF treatment resulted in an upregulation of GFAP expression and morphological differentiation of RPCs. (G, J): Fluorescent images of PKC{alpha} immunoreactivity; (H, K): GFP expression in the same RPCs. (I, L): Combined images of (G, H) and (J, K). CNTF induced RPCs to upregulate PKC{alpha} expression and adopt bipolar morphology. Abbreviations: CNTF, Ciliary neurotrophic factor; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; PKC, protein kinase C; RPC, retinal progenitor cell.

 
Effect of CNTF on Transcription of Bipolar, Glial, and Progenitor Cell Markers
Semiquantitative RT-PCR analysis was carried out to determine the effects of CNTF on late RPC differentiation (Fig. 5Go). RPCs grown in the absence of CNTF (referred to as RPCs in Fig. 5Go) showed clear expression of a range of neurodevelopmental markers, including Hes1, Nestin, Notch1, and Pax6, confirming that the cells maintained their progenitor cell characteristics. After 14 days of CNTF treatment, there was a general decrease in the expression of many of these markers. Nestin expression was downregulated, whereas Hes1, Notch1, and Pax6 expression was almost undetectable. In contrast, there was an increase in the expression of PKC{alpha} and GFAP. The downregulation in transcripts associated with maintaining the progenitor cell state along with a concomitant increase in PKC{alpha} (bipolar) and GFAP (glial) transcripts suggested that CNTF increased the number of RPCs expressing markers of bipolar neurons and glia. However, the absence of mGluR6 (bipolar) expression suggested that the differentiation cues provided by CNTF were insufficient to bring about complete differentiation of RPCs into bipolar neurons. In addition, the complete absence of recoverin and rhodopsin transcripts confirmed the lack of induction of cone bipolar or photoreceptor differentiation by CNTF.



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  		Figure 5. Reverse transcription--polymerase chain reaction analysis of late RPCs grown in the absence or presence of CNTF. Late RPCs grown in the absence of CNTF expressed a range of neurodevelopmental markers, including Nestin (neuronal progenitor cell marker), Notch1 (surface receptor), Hes1, and Pax6 (nuclear transcription factors). Treatment with CNTF for 14 days resulted in decreased expression of Nestin and complete downregulation of Hes1, Notch1, and Pax6. There was concomitant increase in bipolar (PKC{alpha}) and glial (GFAP) cell specific markers. Transcripts for bipolar (mGluR6) and photoreceptor (recoverin and rhodopsin) specific cell markers were not detected in untreated or treated RPCs. Abbreviations: CNTF, Ciliary neurotrophic factor; GFAP, glial fibrillary acidic protein; PKC, protein kinase C; RPC, retinal progenitor cell.

 
Determination of the Effect of CNTF on GFAP, Nestin, PKC{alpha}, and Recoverin Protein Expression Using Immunoblot Analysis
The RT-PCR results were corroborated by immunoblot analysis of late RPCs grown in the absence or presence of CNTF (Fig. 6Go). Late RPCs grown in the absence of CNTF (referred to as RPCs in Fig. 6Go) expressed high levels of Nestin. In addition, a low level of PKC{alpha} protein expression was also detected in these cells at baseline levels. Treatment with CNTF for 14 days resulted in a twofold increase in PKC{alpha} expression and a simultaneous fourfold decrease in Nestin expression. Furthermore, RPCs treated with CNTF showed a strong band indicative of GFAP expression, whereas a very faint band was detected in the control lane (Fig. 6Go). The data from immunoblot analysis suggested that CNTF treatment induced the late RPCs to upregulate bipolar and glial markers. The absence of recoverin expression excluded the possibility of induction of differentiation of RPCs by CNTF into a subtype of cone bipolar cells [21].



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  		Figure 6. Immunoblot analysis of late RPCs grown in the absence or presence of CNTF. Late RPCs, grown in the absence of CNTF, expressed high levels of Nestin. In addition, PKC{alpha} protein expression was also detected in these cells at baseline levels. Treatment with CNTF for 14 days resulted in an upregulation of GFAP and PKC{alpha} expression and a simultaneous downregulation of Nestin expression. However, recoverin expression was not detected in untreated or treated RPCs. Retinas from B6 mice (4 weeks) were used as negative or positive controls. Abbreviations: CNTF, Ciliary neurotrophic factor; GFAP, glial fibrillary acidic protein; PKC, protein kinase C; RPC, retinal progenitor cell.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was undertaken to investigate the effects of CNTF on in vitro differentiation of late retinal progenitor cells. These RPCs are multipotential and have the capacity to differentiate into any of the cell types generated during late retinogenesis [2, 3, 22]. Results from our immunocytochemical, RT-PCR, and/or immunoblot analysis indicated that CNTF induced a large proportion of late RPCs to differentiate into cells that morphologically resembled bipolar neurons and expressed PKC{alpha}. However, the absence of mGluR6 expression suggested that these cells had not undergone complete bipolar differentiation and may require other extrinsic signals to attain their final state of differentiation. In addition to upregulation of bipolar markers, an increase in GFAP expression was also observed in our studies. Further experiments will be needed to identify the GFAP-expressing cells, such as astrocytes or Müller glia, that were generated as a result of CNTF treatment. The possibility that GFAP-expressing cells may not be either astrocytes or Müller glia but immature progenitor cells must also be considered. Previous studies have shown that GFAP expression does not invariably correlate with other markers of astrocytic phenotype or function [23]. In addition, several investigators have reported that neural stem cells either express GFAP or have done so during their ontogenic development [24]. Hence, neural stem and progenitor cells may go through stages where they express GFAP without necessarily being astrocytes at these points in time [25]. Therefore, GFAP should be viewed as an intermediate-filament protein marker of immature progenitor cells as well as mature astrocytes.

In contrast to its effects on bipolar and glial markers, CNTF inhibited or did not induce expression of rod photoreceptor markers in RPCs in the present study. In the past, CNTF has been implicated as a regulator of rod photoreceptor differentiation during a transient period of development in the vertebrate retina, albeit with opposite effects in the chick and rodent retina. In the chick, CNTF has a positive effect on rod photoreceptor differentiation [15, 16], whereas in the rodent retina, CNTF acts as a negative regulator of rod photoreceptor differentiation in vitro [17, 18, 26]. Results from previous studies on the inhibitory effect of CNTF on rod development in the rat retina have been controversial. Experiments in dissociated and organotypic slice cultures of rat retinas demonstrated that CNTF acted as a transient and negative regulator of rod differentiation [17, 26]. In those studies, CNTF inhibited the increase in rhodpsin and recoverin-positive cells, although cells that had already started to express rod-specific markers remained unaffected. This inhibitory effect of CNTF on rod development was not accompanied by compensatory changes in the proportion of other cell types. In contrast, when CNTF was added to postnatal rat retinal cultures, the expression of markers specific to two cell types, rod photoreceptors and bipolar neurons, was dramatically affected [18, 27]. It was concluded from these observations that CNTF altered cell fate by inducing rod progenitors to change lineage and follow the bipolar pathway. Interestingly, although the resulting neurons expressed bipolar markers, they did not resemble bipolar cells and were closer in morphology to the neighboring opsin-positive rods. A slight increase in the percentage of cells expressing GFAP was also noted in this study, attributed to increased survival or proliferation of retinal astrocytes or upregulation of GFAP expression by Müller glial cells [18]. In a recent study, the GFAP promoter was used to drive Müller cell–specific expression of GFP in transgenic mice. The experimental data from this study showed that intravitreal injection of CNTF led to the upregulation of both endogenous GFAP and the GFP transgene in the retina [28]. Hence the experimental findings of the present study are in general accord with the results of previous studies, showing an increase in the number of cells expressing bipolar and glial markers and inhibition of rod photoreceptor differentiation in response to CNTF treatment [18, 27].

Results from RT-PCR analysis performed in the present study showed that CNTF-induced differentiation of late RPCs was accompanied by a simultaneous downregulation of Nestin and undetectable levels of Hes1, Notch1, and Pax1 transcripts. The decrease in Nestin expression was confirmed by immunocytochemical and immunoblot analysis. Results from immunostaining experiments showed that the number of RPCs that stained positive for Nestin decreased from > 86.5% to 48.5%. In addition, the intensity of Nestin staining also decreased in the CNTF-treated RPCs. These observations were in accordance with the following findings made in previous studies.

Nestin [29] is an established marker for neuronal/retinal progenitor cells [19, 30, 31]. It is expressed in progenitor cells in their proliferating and undifferentiated state. When these cells undergo their final mitotic division and begin to differentiate into mature neurons, Nestin expression is downregulated [32].

Notch1, a transmembrane receptor gene, and Hes1, a basic helix-loop-helix transcription factor gene, are expressed in RPCs and downregulated in differentiating and mature neurons [33, 34]. Both of these genes seem to be involved in maintaining RPCs in an undifferentiated state. Previous studies showed that forced expression of a constitutively activated Notch1 gene in rat retinal progenitor cells blocked the normal differentiation of the neuronal cell types and promoted formation of an unidentified cell type [33]. Similarly, the persistent expression of Hes1 blocked retinal cell differentiation [34]. Pax6, a paired box transcription factor, is required for the maintenance of the multipotential state of RPCs [35]. Upon experimental inactivation of Pax6, the potential of RPCs becomes restricted to only one of the fates available to RPCs, namely the amacrine cell type [35]. In summary, results from the present in vitro study with late RPCs were consistent with the findings of previous in vivo studies showing that Hes1, Nestin, Notch1, and Pax1 transcripts are expressed in progenitor cells in the developing retina and downregulated as the cells undergo differentiation.

In the present study, 12.8% of RPCs stained positive for PKC{alpha} in the untreated controls. The PKC{alpha} expression was corroborated by RT-PCR and immunoblot analysis. In addition, 8.2% of RPCs also stained positive for GFAP in the untreated controls. GFAP expression was also detected in late RPCs grown in the absence of CNTF using RT-PCR and immunoblot analysis. Several hypothesis can be put forward to explain the baseline expression of these mature markers in late RPCs. One possibility could be that late RPCs consist of mixed populations of cells that include progenitor cells that express Nestin but no mature cell markers, glial precursor cells that express Nestin and GFAP, bipolar precursor cells that express Nestin and PKC{alpha}, and bipotential precursor cells that express Nestin as well as GFAP and PKC{alpha}. These bipotential cells can differentiate into bipolar neurons or glia in response to the appropriate extrinsic signals by upregulation of the appropriate genes and switching off the others. Our results do not exclude this possibility. Another, perhaps more attractive, hypothesis would be that the baseline expression of mature cell markers in late RPCs is unique to these cells and a byproduct of expansion in culture. These mitotic cells continue to divide, and their numbers expand while they are nonetheless in transit between their parental progenitor cells and their mature progeny. Regardless of the markers expressed by these cells, they seem neither to be functionally mature nor terminally differentiated. This hypothesis is supported by experimental data from previous studies carried out with human embryonic stem cells [36]. Once again, our data do not exclude this possibility.

In the present study, when RPCs were seeded onto poly-D-lysine/mouse laminin-coated eight-well culture slides and treated with CNTF for 14 days, 10% of the total cells survived. This finding suggests that CNTF treatment may result in a positive selection of bipolar and glial precursor cell sub-populations that exist within the total RPC cell population. In contrast, when RPCs were treated with medium containing no EGF and no CNTF for 14 days, < 1% of cells survived (data not shown). From these results it can be concluded that RPCs are delicate cells and that the absence of EGF in the media inevitably leads to cell death of many progenitors. However, addition of serum or differentiation factors such as CNTF to the media in the absence of EGF can support RPC growth in vitro. These findings suggest that CNTF may affect RPC differentiation indirectly by enhancing survival of late RPCs. Alternatively, CNTF may directly induce RPC differentiation by selectively enhancing survival of bipolar and glial precursor cells.

In summary, our results show that CNTF can induce expression of bipolar and glial markers along with a simultaneous downregulation of progenitor and proliferative markers. Upregulation of cell-specific markers was accompanied by morphological differentiation into bipolar and glial phenotypes. However, the absence of mGluR6 expression in PKC{alpha}-expressing cells strongly suggests that additional extrinsic factors are required, not only to completely differentiate RPCs into bipolar cells but also to downregulate GFAP expression.

Finally, having demonstrated the effects of CNTF on bipolar and glial differentiation of expanded late RPCs, we are now working on inducing late RPCs to differentiate into photoreceptors. Once we have developed in vitro differentiation assays for late RPCs that will enable us to control the differentiation of RPCs into photoreceptor as well as bipolar cell types, the next step will be to generate different layers of the retina in vitro by growing these cells on polymer substrates in the presence of different growth factors.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was supported by a kind gift from Richard and Gail Siegal and grants from National Institute of Health NEI 09595 (to M.J.Y.), Laboratory for Drug Discovery in Neurodegeneration (HCNR), Grousbeck Foundation, CHOC Foundation, Minda de Gunzburg Research Center, and Padrinics (to HK). We thank the DSHB, University of Iowa for the Nestin antibody and A. Dizhoor and R. Molday for the Rhodopsin antibody. We thank Marie Shatos for the retinal progenitor cells and Kameran Lashkari for his help with the RT-PCR studies.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 15, 2004; accepted for publication November 19, 2004.



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