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First published online September 22, 2005
Stem Cells Vol. 24 No. 3 March 2006, pp. 696 -706
doi:10.1634/stemcells.2005-0190; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Epidermal Growth Factor Is a Neuronal Differentiation Factor for Retinal Stem Cells In Vitro

Brigitte Angénieuxa,b, Daniel F. Schorderetb,c, Yvan Arsenijevica,b

a Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, Lausanne, Switzerland;
b Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland;
c Institute of Research in Ophthalmology, Sion, Switzerland

Key Words. Radial glia • Cell competence • Retinal ganglion cells • Neurogenesis • Retinal progenitor cell

Correspondence: Yvan Arsenijevic, Ph.D., Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, 15, av. de France, 1004 Lausanne, Switzerland. Telephone: +41-21-626-82-60; Fax: +41-21-626-88.88; e-mail: yvan.arsenijevic{at}ophtal.vd.ch

Received April 25, 2005; accepted for publication September 11, 2005.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stem cells are a tool for in vitro elucidation of the putative role of factors on cell fate. Herein we analyze the role of epidermal growth factor (EGF) on progeny derived from retinal stem cells (RSCs). We isolated cells from neuroretinas of neonate mice. All the proliferating cells harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation 7 days after transfer of the cells in different culture media. In absence of serum, EGF led to the expression of the neuronal marker ß-tubulin-III and acquisition of neuronal morphology in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating cell cycle progression. Moreover, a pulse of 2-hour EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b, and melanopsin) and in a few cases to other retinal phenotypes (photoreceptors [PRs] and bipolar cells). We confirmed that the late RSCs were not restricted over time and that they conserved their multipotency by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
For two decades, great efforts have been made to characterize the in vivo behavior of retinal progenitor cells (RPCs). RPCs, in a precise spatiotemporal evolutionary-conserved manner, produce ganglion cells, horizontal cells, cone photoreceptors, and amacrine cells during early embryogenesis (from embryonic day 10 [E10] to E16 in mice) and rod photoreceptors, bipolar cells, and Müller glia during later embryogenesis and the first postnatal days (from E18 to postnatal day 12 [PN12] in mice) [1–5]. Several studies have shown that RPCs are multipotent [6–8]: in vivo lineage analysis reveals that one RPC can give rise to different retinal cell types. Many groups have focused on the cell fate acquisition of RPCs. For instance, Lillien et al. showed that RPCs are biased toward the glial fate at later stages of retinal development [9]. Several works showed that, in vitro, RPCs reproduce the in vivo cell generation: early RPCs (isolated at E14) generate early-born neurons, whereas late RPCs (isolated after E16) generate late-born neurons. In most studies, late RPCs were analyzed only after their isolation or after limited expansion [10–14]. Therefore, the issue of their expansion potential, as well as the conservation of multipotency over cell passages and time (i.e., defining their stemness potential), remains to be addressed.

During embryogenesis, the retina derives from the forebrain and thus belongs to the central nervous system (CNS). The potential of brain neural stem/progenitor cells to generate specific cell types in vitro has been well studied and well characterized. The behavior of retinal cells has been studied mostly in vivo. To be able to culture retinal progenitor/stem cells and instruct them to generate different cell types would help to dissect the gene expression profile in different conditions or the factors involved in cell fate determination, as was previously done with neural stem cells (NSCs) to elucidate the differentiation of GABAergic neurons [15, 16] or cholinergic neurons [17]. These experiments should help to clarify which cues support or drive the induction of a specific cell fate. How intrinsic versus extrinsic factors govern these differences in cell fate potential during development is an intensively studied question. Different extrinsic factors are known to control retinal cell fate specification. For example, fibroblast growth factor (FGF)-2 (reviewed in [18]), taurine [19], and sonic hedgehog [20] control photoreceptor development, whereas epidermal growth factor (EGF) promotes RPC proliferation or differentiation toward the astrocytic phenotype cell fate [21–23].

To further elucidate the role of extrinsic factors on retinal cell fate after in vitro expansion, we investigated whether late proliferating cells present at birth meet the definition of retinal stem cells (RSCs) and are able to generate different retinal cell types after stimulation by a single growth factor. We also challenged their ability to generate early retinal neurons after expansion in order to reveal whether they lose their potentiality in proportion to division number or maintain the potency to generate different types of retinal neurons throughout passages. We showed that EGF can act as a neuronal differentiating factor. Our results also revealed that the postnatal eye contains RSCs in the radial glia cell population that can be passed in vitro over a long period of time, expanded extensively, and induced to differentiate toward diverse neural phenotypes (glial cells, retinal ganglion cells, and photoreceptor cells) in the sole presence of EGF and an adhesive substrate. Moreover, RSCs can be used to screen the effects of various factors on retinal differentiation and to determine the genes involved in this mechanism.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Dissection Procedure
All animal procedures were allowed by the Veterinary Service of the State of Vaud, Switzerland. We dissected eyes from strains of DBA/2 or OKABE postnatal mice (between passage 0 [P0] and P2). The eyes were placed in oxygenated artificial cerebral spinal fluid (aCSF; 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 26 mM NaHCO3, and 10 mM D-glucose; Sigma, Buchs, Switzerland, http://www.sigmaaldrich.com) in a separate Petri dish. The optic nerve, the retinal pigmented epithelium, and the central retina were carefully removed. The dissected retina was then digested in a trypsin mix (aCSF modified to contain high Mg2+[3.2 mM MgCl2 ] and low Ca2+[0.1 mMCaCl2], 1.33 mg/ml trypsin, 0.67 mg/ml hyaluronidase, and 0.2 mg/ml kynerunic acid). All products were purchased from Sigma. After 10 minutes at 37°C, 0.1 mg of trypsin inhibitor (ovamucoid; Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com) was added to the solution and cells were triturated 20 to 30 times. The complete solution was then centrifuged at 1,800 rpm, and the enzyme solution was removed and replaced by serum-free medium [24] containing 20 ng/ml EGF (Peprotech, Rocky Hill, NJ, http://www.peprotech.com) and 20 ng/ml basic FGF-2 (Peprotech). The cells were seeded in a 75-cm2 flask (Nunclon, Naperville, IL, http://www.nuncbrand.com) at a density of either 3 or 5 x 105 cells per ml.

Expansion of RSCs
After dissection, single cells formed small colonies and then gave rise to a monolayer culture within 1 month. The cells grew in EGF and FGF-2 (20 ng/ml each). Media was changed every 3 days. When cells reached approximately 100% confluence, they were trypsinized and either expanded or induced to differentiate. For expansion, the cells were plated in a new 75-cm2 flask at a density of either 2 or 4 x 105 cell per ml in serum-free medium containing FGF-2 and EGF (20 ng/ml each).

Differentiation
To induce differentiation, we seeded 40,000 single cells onto glass coverslips coated with poly-L-ornithin with laminin placed in 24-well plates (well size, 2 cm2; Nunclon). We induced differentiation by adding different factors and analyzed the cells by immunocytochemistry after 1, 7, or 10 days. For the conditions of differentiation, see Figure 1Go. Briefly, to discriminate between the mitogenic or the inducing effect of EGF, we carried out short-term or long-term EGF stimulations. After 7 days, we either extracted the RNA or fixed the cells to perform immunocytochemistry. To analyze the multipotent characteristic, we transferred 10 OKABE cells (green fluorescent protein-positive [GFP+]) mixed with 40,000 cells (GFP+).


Figure 1
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  		Figure 1. Protocol for retinal stem cell (RSC) differentiation. The cells were expanded (expansion part) or induced to differentiate at confluence. After dissociation, single cells were plated onto coverslips coated with polyornithin and laminin in a medium containing EGF (20 ng/ml). After 2 hours, the medium was removed and replaced with either fresh medium alone (short-term experiments) or fresh medium containing EGF (control, long-term experiments). After 7 days, RNA was extracted or the cells were fixed for immunochemistry analysis. Another control consisted of a constant exposure to EGF. To analyze the multipotency of the RSCs after several passages, we plated 10 cells expressing the GFP transgene, mixed with 40,000 DBA/2J cells. After a two-step differentiation protocol (10 DIV), we fixed the cells and performed immunostainings to characterize neuronal and glial cells. Abbreviations: EGF, epidermal growth factor; GFP, green fluorescent protein; wt, wild-type; DIV, days in vitro; bFGF, basic fibroblast growth factor; FGF, fibroblast growth factor; P34, passage 34.

 
To analyze the direct action of EGF, we plated the cells at diverse densities (500 or 1,000 cells per well). We induced differentiation as previously described. We fixed the cells after 7 days and performed immunocytochemistry.

Bromodeoxyuridine Labeling
After plating the cells onto coverslips, we added 500 nM bromodeoxyuridine (BrdU; Sigma) to the medium. In the short-term and long-term experiments, the fresh media also contained 500 nM BrdU. In accordance with manufacturer instructions (anti-BrdU and Nuclease detection kit; Amersham Biosciences, Buckinghamshire, U.K., http://www.amersham.com), we revealed the labeling after fixation of the cells with 4% paraformaldehyde.

Immunocytochemistry
To characterize the RSCs and determine cell differentiation, we performed immunocytochemistry analyses. We sequentially fixed the cells with 4% paraformaldehyde for 20 minutes at room temperature and incubated them with primary antibodies and then with fluorescent-conjugated secondary antibodies for 1 hour at room temperature. Primary antibody concentrations and times of incubation were as follows: overnight at 4°C for anti-nestin rabbit polyclonal (1:500; gift from Dr. R.D.G. McKay), anti-melanopsin rabbit polyclonal (1:100; Abcam, Cambridge, U.K., http://www.abcam.com), and anti-Bmi1 mouse monoclonal [25] (1:40; gift from Dr. van Lohuizen), and 2 hours at 37°C for anti-ß-tubulin-III mouse monoclonal (1:1000, Sigma), anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal (1:400; Sigma), anti-RC2 mouse monoclonal (1:4; Santa Cruz Biotechnology, Inc., LabForce AG, Nunningen, Switzerland, http://www.labforce.ch), and anti-recoverin rabbit polyclonal (1:500; Sigma). Secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) were as follows: cyanine-conjugated affinity-purified goat antibody to mouse immunoglobulin (Ig) G (1:1000), fluorescein isothiocyanate (FITC)-conjugated affinity-purified goat antibody to mouse IgG (1:100), and 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated affinity-purified goat antibody to mouse IgM (1:100). After immunostaining, we counterla-beled all cells with Hoechst 33258 nuclear staining (1:333; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). For each experiment, a total of 1000 cells were counted.

mRNA Analysis
Total RNA was extracted using the TRIZOL reagent (Invitrogen, Life Technologies, Basel, Switzerland, http://www.invitrogen.com). Reverse transcription (RT) was performed from 1 µg of total RNA, using random primers from Promega (Madison, WI, http://www.promega.com). Polymerase chain reaction (PCR) reactions were incubated for 5 minutes at 94°C, then for 30 cycles at 94°C for 30 seconds, at melting temperature (Tm) for 1 minute, and at 72°C for 1 minute followed by a final elongation time of 10 minutes at 72°C using the following primers: Brn3b sense primer 5'-AATGAATTCATCCACGTCGCTCATGCAG-3' and anti-sense primer 5'-AATGTCGACCTGAGCGTAATGTGTGCCTTC-3', Tm = 68°C, and Pax6 sense primer 5'-GGGCAGGTATTACGAGACTGG-3' and antisense primer 5'-GAGACAGGTGTGGTGGGCTG-3', Tm = 65°C. Touchdown PCRs were performed using the following primers: for Math5 sense primer 5'-TGGATGAAGTCGGCCTGCAA-3' and Math5 antisense primer 5'-TGGATGAAGTCGGCCTGCAA-3', for glyseraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer 5'-ACCACAGTCCATGCCATC AC-3' and GAPDH antisense primer 5'-TCCACCACCCTGTTGCTGTA-3' and for Mash1 sense primer 5'-TTGAACTCTATGGCGGGTTC-3' and antisense primer 5'-GGTTGGCTGTCTGGT TTGTT-3'.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Postnatal Retina Contains RSCs That Can Be Easily Expanded In Vitro
All retinal cell types are produced during embryogenesis in a precise and irreversible order [3, 5]. The peak of photoreceptor production is reached in approximately the first postnatal days. Thus, at this period the retina contains highly proliferative cells, and we expected to isolate highly expandable RSCs. After dissection (see Materials and Methods) and dissociation, we plated the single-cell suspension in serum-free media containing FGF-2 and EGF. Within the first days, most of the cells died. The remaining cells grew in clusters, leading in less than 1 month to a monolayer culture of cells with a homogenous morphology (Fig. 2A, 2BGo). Then these cells expanded easily in vitro as shown by their high potency to proliferate. Five million cells (number of cells plated at P0) could generate more than 1046 cells (P34) in a period of almost 1 year (cells are still growing; Fig. 2CGo). At early and late expansion stages (P1–P34), the removal of growth factors led to cell death, demonstrating that the cells had not undergone transformation, a phenomenon that can occur during NSC expansion [26]. Moreover, after subretinal transplantations in a degenerating retina, we observed no tumor formation even after 3 months (n = 3; K. Canola, personal communication). One characteristic of the stem cell is the maintenance of its undifferentiated state. The cultured cells expressed the nestin filament [27] (96 ± 1.2%, n = 4; Fig. 2DGo), characteristic of progenitor cells, and Bmi1 [25, 28, 29] (100%, n = 4; Fig. 2EGo), a gene required for neural stem proliferation. Interestingly, all the cells also expressed RC2 (100%, n = 4; Fig. 3DGo), a marker of radial glia that contain the postnatal NSCs in their population [30]. RC2 is the most commonly used radial glia marker [30 –34] because it is present throughout radial glia differentiation and maturation [30]. In the brain, NSCs are present in the RC2-positive population during embryogenesis, then in astrocytes derived from the radial glia [32]. Using immunocytochemistry, we controlled the presence of RC2-positive cells in the retina of newborn (Fig. 3A, 3BGo) and adult (Fig. 3CGo) mice. Only the postnatal retina shows RC2 labeling (Fig. 3Go). The cultured cells also expressed Mash1 and Pax6 (n = 3; Fig. 3EGo), two transcription factors that have been shown to be expressed by RSCs and radial glia [11, 34]. No markers of the differentiated state, such as GFAP or ß-tubulin-III, were observed during the growth process (data not shown). Consequently, our culture procedure allowed the expansion of only a population of radial glia cells which harbors RSCs. To prove their multipotency, we then showed that after expansion, one single cell could give rise to neurons and glia cells. To expand clonally and to improve differentiation of clones derived from RSC cultures, we mixed 10 GFP-expressing cells with 40,000 cells. We investigated the cells at late passage (P14). We analyzed the expression of GFAP (a glial marker) and ß-tubulin-III (a neuronal marker) in GFP+ cells after differentiation. The cells were first cultured for 5 days in the presence of FGF-2 and heparin sulfate followed by another 5 days with the B27 supplement. Out of 32 coverslips analyzed, one to 10 green cells or groups of green cells per coverslip were found, attesting to the correctness of the dilution process. In total, 148 colonies were detected when at most 320 were expected: 45.3% of the clones contained only neurons, 15.5% only glial cells, and 39.2% were composed of neurons and glia (Fig. 4Go). Five point four percent of the GFP+ plated cells gave rise to large colonies containing the two cell lineages (25– 85 cells per clone). Thus, the cells conserved their multipotency and capacity to generate large clones even after numerous passages (P14), two essential characteristics of stem cells. It appeared that our culture conditions allowed the propagation of RSCs.


Figure 2
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  		Figure 2. The mouse postnatal retina contains highly proliferative RSCs. After dissection and dissociation, the cell suspension was plated in a medium containing epidermal growth factor (EGF) (20 ng/ml) and fibroblast growth factor (FGF)-2 (20 ng/ml). After a few days, small clusters of cells proliferated (A), leading to a monolayer culture (B) within 1 month. The cells showed a high expansion potential; 5 x 106 cells could generate more than 1046 cells (C) in less than 1 year. The cells are still growing. We have not pushed all the cell cultures (n = 5) to their limit of expansion; however, cell cultures (named B, C, F, K, and T) showed the same shape of expansion curve. Note that RSC F' corresponds to the thawing of RSC F culture. The scale is logarithmic. The removal of growth factors arrested proliferation. To confirm that we expanded undifferentiated cells, we plated them on an adhesive substrate. Twenty-four hours later, we fixed the cells and analyzed the presence of two markers expressed in neural stem/progenitor cells. (D): Ninety-eight percent of the RSCs expressed the nestin filament. (E): All of them expressed the transcription factor Bmi-1, a gene required for the maintenance of the neural precursor state. Note the nuclear staining of Bmi-1. Magnification: x100 (A, B), x200 (D), and x400 (E). Abbreviations: RSC, retinal stem cell; DAPI, 4',6-diamidino-2-phenylindole.

 

Figure 3
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  		Figure 3. Retinal stem cells (RSCs) harbored radial glial characteristics. (A, B): In postnatal day 0 through 1 retinas, numerous cells in the inner part of the retina expressed the canonical radial glial marker RC2. Arrows point to representative RC2-positive cells with their visible nucleus and cell body. (B): Few cells highly expressed RC2. (C): No expression of RC2 was detected in the adult retina. (D): All cultured cells also expressed the radial glia marker RC2. (E): Reverse transcription-polymerase chain reaction analysis revealed that the RSCs expressed Pax6 and Mash1. Magnification: x1000 (A, B), x400 (C).

 

Figure 4
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  		Figure 4. The highly proliferative cells are multipotent. One single cell can expand and differentiate into the two cell lineages of the retina. We induced differentiation as described in Figure 1Go (clonal analysis) and analyzed the expression of ß-tubulin-III and GFAP in clonally expanded GFP-expressing cells. We observed that approximately 5% (see Results) of the clones had generated neuronal cells as well as glial cells (bipotent clones), whereas other clones had generated either glial cells or neurons only (unipotent clone; data not shown). (A): ß-Tubulin-III labeling (red) of wild-type and of a single GFP+ colony derived from one clone (C). (B): GFAP staining (blue) of the same clone and of wild-type cells. (C): GFP fluorescence (green) of the clone. (D): Merged picture of (A–C). White arrows show the neurons derived from the GFP+-retinal stem cell (RSC) clone, and yellow arrows indicate a glia cell derived from GFP+-RSCs. (E): Enlargement of the superposition of three stainings, marked by a white square in (A–C). Magnification: x200 (A–D). Abbreviations: GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.

 
EGF Promotes Neuronal Formation
After cell expansion, we tested several factors to investigate whether a specific factor could induce RSC differentiation toward a specific cell phenotype. To induce differentiation, we transferred RSCs from early (P4) to late (P21) passages onto laminin-coated coverslips and tested different media to direct the cells toward the neuronal or glial lineage. We focused on EGF- and FGF-2–containing media because EGF was shown to induce the formation of glia during brain development [35] or in vitro [9] whereas FGF-2 was observed to promote the neuronal lineage [36–38]. We tested EGF, FGF-2, and fetal bovine serum (FBS) either alone or in combination during an incubation period of 7 days. In absence of growth factors or serum, RSCs and the derived progeny did not survive, whereas the number of ß-tubulin-III–positive cells increased in the sole presence of EGF (Fig. 5AGo). Cells expressing ß-tubulin-III showed a neuronal morphology and represented 15.35 ± 3.7% (n = 4) of the total cell population. We also observed an increased number of neurons with FGF-2, but we focused the present study on the unusual action of EGF on neuron formation. Interestingly, when 2% FBS was added to EGF-containing medium, we observed an almost complete absence of ß-tubulin-III–positive cells and their replacement by cells harboring a glial morphology with a large nucleus and expressing the glial marker GFAP (Fig. 5BGo). This showed that EGF had the ability to increase the formation of neurons from RSCs and that serum prevented this action.


Figure 5
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  		Figure 5. EGF promotes neuronal formation. (A): We tested different epigenetic factors on neuronal formation. EGF long- (7 DIV) and short-term exposure (EGF, 2 hours) generated the higher percentage of ß-tubulin-III–positive cells. The generation of ß-tubulin-III–expressing cells was almost abolished with the addition of FBS (FBS + EGF) and dramatically weakened in the presence of FGF-2 or in the presence of both EGF and FGF-2. (B): In the presence of serum, most cells expressed the glial marker GFAP. Magnification: x200. Abbreviations: EGF, epidermal growth factor; DIV, days in vitro; FBS, fetal bovine serum; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; DAPI, 4',6-diamidino-2-phenylindole.

 
EGF Induces Neuronal Differentiation of RSCs
We next wanted to analyze the effect of EGF on neurons. Thus, we carried out different experiments to determine whether EGF acts as a mitogen, a survival, or a differentiating factor. These different possible actions were investigated in the experiments described below. The presence of an increased number of cells with a neuronal phenotype can be due to divisions of progenitors committed to the neuronal fate. EGF is indeed known mostly for its mitogenic property in vivo [24, 39, 40]. In our in vitro experiments, EGF could thus act by inducing the proliferation of neuroblasts that afterward differentiated into ß-tubulin-III–positive cells. To investigate this hypothesis and follow the cells that had undergone division, we added BrdU throughout the differentiation process. After 7 days in vitro, only 21.49 ± 7.71% (n = 3) of the ß-tubulin-III–positive cells were also BrdU+ (Fig. 6Go). This low percentage revealed that a large number of neurons formed in vitro under EGF stimulation had not undergone cell proliferation prior to differentiation. Thus, most of the neurons were not formed under EGF stimulation as a result of the proliferation of retinal neuroblasts in these culture conditions. To confirm that EGF did not act as a mitogen on these cells, we exposed them to short or long pulses of EGF. An effect after a short exposure would indicate that EGF triggers a differentiating program as previously shown for insulin-like growth factor (IGF)-1[41] or platelet-derived growth factor (PDGF)-{alpha}[42], whereas an effect after long exposure would reveal that EGF was involved in the proliferation process since the presence of a mitogen is required to maintain proliferation. It appeared that a short exposure to EGF was sufficient to induce neuronal differentiation. Indeed, 2 hours after the transfer of the cells onto a laminin substrate in the presence of EGF, we removed the medium and replaced it either with fresh medium alone (short-term exposure) or with fresh medium containing EGF (long-term exposure). In both cases, we observed that 15% of the cells expressed ß-tubulin-III (15.83 ± 3.92% for the short exposure and 15.85 ± 3.7% for the long exposure [n = 4]; Fig. 5AGo). Thus, 2-hour EGF exposure was sufficient to induce the formation of neurons. However, the cells did not present the same pattern of ß-tubulin-III expression nor the same morphology as after sustained exposure. In the short-exposure experiments, the ß-tubulin-III–positive cells were immature with a round cell body and very few short neurites (Fig. 7AGo), whereas with sustained exposure, the cells acquired a more mature phenotype with long and fine processes (Fig. 7BGo). These data, taken together with the BrdU experiments, showed that EGF did not induce neuron production through stimulation of cell division.


Figure 6
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  		Figure 6. The neurons formed in the presence of epidermal growth factor (EGF) incorporated BrdU poorly. To reveal whether neuroblast proliferation occurred during EGF exposure, BrdU (500 nM) was added throughout the differentiation process. After 7 days, only 20% of the ß-tubulin-III–positive cells were positive for BrdU+. (A): The ß-tubulin-III–positive cell (green, encircled) is BrdU-positive (red), meaning that during EGF exposure, the cell divided and differentiated toward the neuronal fate. The other ß-tubulin-III–positive cell (square) is BrdU-negative, meaning that division is not required for differentiation. Note that the three other ß-tubulin-III–positive cells (arrows) did not incorporate BrdU. Some other cells (not labeled by ß-tubulin-III) were also BrdU-positive (arrowhead). For full quantification, see Results. (B): High magnification of the nuclei circled in (A). Blue fluorescence represents Hoechst-stained nuclei. Magnification: x200. Abbreviation: BrdU, bromodeoxyuridine.

 

Figure 7
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  		Figure 7. EGF initiates a neural program. (A): Short exposure to EGF led to immature neurons. ß-Tubulin-III was expressed mostly around the cellular body (arrowhead). On a few cells, processes started to grow (arrows), indicating the beginning of neuronal maturation. (B): Long-term exposure to EGF sustained neuronal maturation, as revealed by the appearance of long and fine neurites. (C): After long-term exposure (EGF 7 DIV), the cells expressed the transcription factor Brn3b, a specific marker of retinal ganglion cells, and the transcription factor Ath5, expressed in ganglion cells. The "c" notation refers to a reaction without reverse transcription. (D): In rare cases, EGF led to the formation of recoverin-positive cells (white arrows), which are expressed by photoreceptors and some rare bipolar cells. (E): Enlargement of a recoverin-positive cell (central arrow). (F, G): Long-term exposure to EGF to induce the formation of ß-tubulin-III–positive cells (F) that are also melanopsin-positive (G). Arrows show a double-positive cell (ß-tubulin-III+-melanopsin+ cell), whereas arrowheads show a ß-tubulin-III–positive cell that did not express melanopsin. Note that some cells express melanopsin at a very low level. Magnification: x200 (A, B), x400 (D, F, G). Abbreviations: EGF, epidermal growth factor; DIV, days in vitro.

 
Alternately, EGF could act as a survival factor for the neurons produced during expansion or differentiation. We observed no ß-tubulin-III–positive cells prior to differentiation, and short EGF exposures generated the same number of ß-tubulin-III–positive cells as did long-exposure experiments. These results indicated that the increased neuron number observed in the presence of EGF was not due to a survival action of EGF.

Thus, EGF induced a neuronal differentiation process (i.e., the passage of a nondifferentiated state toward a neuronal phenotype). To determine whether the action of EGF was direct or indirect, cells were plated at low density (500–1,000 cells per well) to prevent cell interactions and paracrine effects. In these culture conditions, differentiation can be attributed only to the cell itself and not to a contribution from adjacent cell(s). We observed that at low density, 29.5 ± 7.24% (n = 3) of the cells were ß-tubulin-III–positive after EGF stimulation. However, the cells were less mature, much as they were after short exposures to EGF, than those in the high-density cultures. In the low-density cultures, the ß-tubulin-III–positive cells appeared immature with few and short neurites (same morphology as shown in Figure 7AGo), whereas in the high-density cultures, the cells expressing ß-tubulin-III acquired a much more mature phenotype with long and fine ß-tubulin-III+ processes (similar morphology as shown in Figure 7BGo). Thus, EGF alone was sufficient to start the differentiation program, but cell–cell contacts as well as the sustained presence of EGF seem to be necessary for maintaining the neuronal program and for the cells to progress along the maturation process.

Cells Differentiating Toward the Neuronal Phenotype Express Markers of Retinal Neurons
We found that EGF increased the number of ß-tubulin-III–positive cells in a direct manner, but the specificity of the neuronal phenotype remained unknown. The ß-tubulin-III–positive cells had long and fine processes, leading us to suppose that some of the newly formed neurons were retinal ganglion cells. This is further corroborated by the fact that only retinal ganglion cells express ß-tubulin-III in the mature retina.

To confirm that EGF induced a retinal ganglion cell fate, we analyzed the expression of specific transcription factors for retinal ganglion cells at the RNA level. RT-PCR experiments in cells differentiated in the presence of long-term EGF exposure showed that these cells expressed Math5 (n = 4; Fig. 7CGo), a transcription factor required for retinal ganglion cell differentiation and expressed during the first steps of retinogenesis [43]. The expression was detected after a sustained exposure (n = 3) and not after short exposure. We also detected Math5 expression after the end of cell passaging, indicating that this transcription factor is also expressed in RPCs as previously described [43]. This confirmed that sustained exposure to EGF was required to maintain Math5 expression. Because Math5 is necessary for RGC differentiation but not sufficient, we also investigated the presence of Brn3b, a factor involved in a later stage of ganglion cell differentiation, which is required for further maturation of ganglion cells [44]. Brn3b was not detected during cell expansion or after short EGF exposure. It was detected (also by RT-PCR) only after sustained exposure to EGF (n = 2; Fig. 7CGo), indicating that some cells are committed to the retinal ganglion cell fate. Furthermore, we also detected numerous ß-tubulin-III–positive cells expressing melanospin, a protein specifically expressed by retinal ganglion cell (Fig. 7F, 7GGo). In rare cases, immunostaining revealed the presence of recoverin-positive cells, which are expressed by photoreceptors and some rare bipolar cells (Fig. 7D, 7EGo). We were not able to detect cells positive for syntaxin (amacrine cells), protein kinase C (PKC) (bipolar cells), or rhodopsin (photoreceptors). Taken together, these results demonstrate that EGF can induce the differentiation of certain early and late retinal neurons from expanded RSCs although other factors are needed for a complete maturation of these neurons.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we showed that postnatal neuroretinas contained RSCs that are part of the radial glia population. We also demonstrated that EGF was not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro.

We have isolated and propagated cells that all expressed the radial glia marker RC2. The cells in our cultures meet the criteria of RSCs: expansion, undifferentiated state throughout passages, and clonal differentiation into the main cell types composing the retina (neurons and glia). Besides, the RSCs expressed the radial glia marker RC2 in addition to Mash1 and Pax6. Several groups have shown that postnatal NSCs are a subpopulation of radial glia that express RC2. Hartfuss et al. [30] have demonstrated that the radial glia also express Mash1. On the other hand, Gotz et al. [34] have shown that Pax6 is required for radial glia formation. We also observed that the retina at the PN0 through PN1 stage highly expressed RC2. This suggested that the culture condition used in this study has selected the RC2-positive cells and that the postnatal RSCs were therefore contained in the radial glia cell population. Likewise, it appeared that retinal cells shared features with postnatal NSCs.

We demonstrated that EGF acted neither as a mitogenic factor for neuroblasts nor as a survival factor for neurons. We first excluded a mitogenic role because few ß-tubulin-III cells incorporated BrdU. Second, short- or long-term exposures produced the same percentage of ß-tubulin-III–positive cells. This experiment also ruled out the hypothesis of a survival effect of EGF. Indeed, if EGF acted as a survival factor, its presence would be required the entire time and its withdrawal would cause the loss of ß-tubulin-III–expressing cells. We did not observe such a phenomenon. On the other hand, we did not observe ß-tubulin-III–positive cells prior to differentiation, meaning that EGF did not allow survival of neurons generated during expansion. These results argue against a survival role for EGF. But one could argue that EGF only amplified the remaining ganglion and/or photoreceptor precursors isolated during the dissection procedure since we did not select a pure population of cells (bulk plating after dissection). The generations of different retinal cell types overlap each other, and at PN0, the vast majority of RPCs are engaged toward the photoreceptor fate and also the beginning of Müller cell production [2–5]. The cells used in this study were able to produce the two main lineages (neurons and glia) of the retina, meaning that they were not precursor cells engaged toward a particular cell phenotype.

The finding that EGF directly induced neuronal differentiation of RSCs and the derived progeny without stimulating proliferation could appear surprising. EGF is known as an important factor for the proliferation of neural stem and progenitor cells in vitro [24, 45] and in vivo, leading to the generation of neurons, astrocytes, and oligodendrocytes [39, 40]. EGF is thought to participate mainly in astrocyte generation. The injection of EGF into the adult rat lateral ventricle or striatum led to an increase of the astrocyte population [40, 46]. Furthermore, a switch from neuron generation to glia cell formation was observed when the EGF receptor was overexpressed [47], showing that the EGF pathway was involved in the glial cell fate decision. On the other hand, EGF can also interfere with neuronal differentiation. The addition of the EGF receptor ligand in rat forebrain cultures inhibited the expression of cholinergic markers [48, 49]. EGF has also been implicated in the survival of NSCs: Loo et al. demonstrated that EGF withdrawal led to the apoptosis of NSCs within 24 hours [50]. Thus, it appeared that EGF had pleiotropic actions in the CNS: stimulation of proliferation, survival action, and induction of astrocyte generation, and our results showed that EGF could also play a role in neuronal differentiation, but this action remains to be determined in vivo.

Because the retina is part of the CNS, various actions of EGF can be expected. In the retina, the EGF receptor starts to be expressed at E15 and reaches its peak of expression during the first postnatal days [9, 22, 51]. These observations suggest the implication of EGF signaling during retinogenesis. Because the highest concentration of the EGF receptor was found at the time we chose to isolate the progenitor cells [9, 22, 51], the implication of EGF in cell generation was not surprising. Such proliferation of EGF on RPC cultures was also reported by Anchan and his colleagues [22]. Moreover, Ahmad et al. [21] have shown that the addition of EGF to E18 retinal explants promoted the proliferation of cells whereas its withdrawal promoted rod differentiation. On the other hand, different groups have revealed that overexpression of EGF receptors promoted glia differentiation from RPCs in culture [9, 51]. EGF can also modulate the differentiation of retinal neurons: the addition of EGF in vitro inhibited rod differentiation and promoted the proliferation of RPCs [22, 51, 52]. Moreover, Fischer and Reh have shown that injection of EGF in combination with insulin into the postnatal chick eye promotes the proliferation of cells located in the ciliary margin zone followed by neuronal differentiation [23]. It appears that EGF, like other factors such as FGF-2, PDGF-{alpha}, or IGF-1 [40, 53, 54], can perform diverse actions on neural stem/progenitor cells. Considering this new insight in vitro, we would find it interesting to elucidate the in vivo action of EGF and transforming growth factor (TGF)-{alpha}, another ligand of the EGF receptor, with regard to the differentiation of retinal cells during development.

During retinogenesis, the generation of postmitotic cells follows a precise spatiotemporal order. Previous studies have suggested that the progenitor cell potential is restricted over time: RPCs isolated early can produce early neurons, whereas late RPCs can generate only late-born neurons. Two types of signals can account for such specification: extracellular signals and intrinsic factors. One model proposes that the progenitor cells go through sequential waves of competence. At a particular time, the progenitor is competent to produce only a subset of a specific cell type. This restriction is intrinsically determined [7, 55] (reviewed in [56]). The progenitor cells are influenced by positive and negative extrinsic signals [7]. An alternative model postulates that the progenitor cells are able to generate all the retinal cells at an early stage but lose this capacity later on. In this model, the progenitor is also intrinsically restricted over time [57]. Thus, these two models predict that, whatever the environment, a progenitor cell isolated at a precise time is restricted to generate certain specific cell types. However, the study of James et al. [58], as well as our work, demonstrates that late progenitor cells placed in a specific environment are still able to respond to early environmental cues and generate earlier-born cells. Thus, the restriction of competence can be overcome in vitro by specific external signals. In our study, we identified EGF as a differentiating factor that induced an early retinal neuronal fate from expanded RSCs that originally were committed to generate photoreceptors and glia. Such reprogramming was also observed with FGF-2 for multipotent progenitors located in the adult optic nerve, an area restricted to generate only glial cells in vivo [59]. Interestingly, in vivo injection of EGF into the newborn chick eye induced the RPCs to generate earlier retinal cell types such as ganglion cells [23]. It appears that in vivo extracellular factors allow the progenitor to differentiate into earlier retinal cell types. Even if the role of EGF on neuronal differentiation during development remains to be determined, its novel action in vitro may be relevant for further experiments to generate specific cell types in vitro and in vivo. This could help to dissect the genes or factors involved in the restriction or specification of retinal cell fate. Moreover, we developed a system to easily expand and culture RSCs which can be used to screen drugs or factors involved in the survival or generation of retinal cells.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We thank R.D.G. McKay and M. van Lohuizen for the generous gift of the anti-nestin and anti-Bmi1 antibodies, respectively. We also thank D. Hornfeld, M. Jaquet, and M. Tekaya for technical help. Special thanks to D. Hornfeld for reviewing the manuscript. This work was supported by the Swiss National Science Foundation, the ProVisu Foundation, the Velux Foundation, and the French Association Against Myopathies.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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