Effects of Ciliary Neurotrophic Factor on Differentiation of Late Retinal Progenitor Cells
http://www.100md.com
《干细胞学杂志》
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@vision.eri.harvard.edu
ABSTRACT
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 . 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 .
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 , glial cell line–derived growth factor , retinoic acid , taurine , and sonic hedgehog , 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 , to inhibit the proliferation of sympathetic precursor cells and induce a change from adrenergic to cholinergic neurons , and to promote differentiation of glial progenitor cells into astrocytes and oligodendrocytes . 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 ? and gp130), which are also components of other cytokine receptors, and the CNTF-specific ligand-binding .
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 . 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 . 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 and mGluR6) in rod precursors located in the photoreceptor layer . 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 .
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
Expression of Retinal Progenitor Markers by Late RPCs
The GFP+ neurospheres (Fig. 1A) 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. 1B). 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 3; Fig. 2A), whereas > 86.5% expressed Nestin (Table 3; Fig. 2B), 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. 2C) and Nestin (Fig. 2D) was downregulated by adherent RPCs upon differentiation.
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.
Table 3. Quantitative evaluation of effect of CNTF on the expression of different cell markers
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. 3A). 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 3). Many of these cells adopted a bipolar morphology and extended long thin neurite-like processes, forming a network between neighboring cells (Fig. 3B).
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 and GFAP expression was determined by immunocytochemistry (Table 3; Fig. 4). After 14 days of CNTF treatment, the percentage of RPCs expressing PKC increased from 12.8% to 41.1% (Table 3). Most of the PKC-positive cells in the CNTF-treated cultures exhibited bipolar morphology (Figs. 4J–4L), whereas those in the untreated cultures remained undifferentiated (Figs. 4G–4I). CNTF also had an inductive effect on GFAP expression. The percentage of cells expressing GFAP increased significantly, from 8.2% to 48.3% (Table 3). The GFAP-positive cells in RPC cultures grown in the absence of CNTF appeared undifferentiated (Figs. 4A–4C), whereas those grown in the presence of CNTF displayed various morphologies, including cells extending many thin, neurite-like processes (Figs. 4D–4F). 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 3). In addition, RPCs grown in the absence of CNTF (Fig. 2B) displayed more intense Nestin staining compared with those grown in the presence of CNTF (Fig. 2D).
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 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 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 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. 5). RPCs grown in the absence of CNTF (referred to as RPCs in Fig. 5) 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 and GFAP. The downregulation in transcripts associated with maintaining the progenitor cell state along with a concomitant increase in PKC (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.
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) 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, 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. 6). Late RPCs grown in the absence of CNTF (referred to as RPCs in Fig. 6) expressed high levels of Nestin. In addition, a low level of PKC protein expression was also detected in these cells at baseline levels. Treatment with CNTF for 14 days resulted in a twofold increase in PKC 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. 6). 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 .
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 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 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
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.
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Turner DL, Cepko CL. A common progenitor for neurons and glia persists in rat retina late in development. Nature 1987;328:131–136.
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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@vision.eri.harvard.edu
ABSTRACT
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 . 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 .
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 , glial cell line–derived growth factor , retinoic acid , taurine , and sonic hedgehog , 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 , to inhibit the proliferation of sympathetic precursor cells and induce a change from adrenergic to cholinergic neurons , and to promote differentiation of glial progenitor cells into astrocytes and oligodendrocytes . 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 ? and gp130), which are also components of other cytokine receptors, and the CNTF-specific ligand-binding .
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 . 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 . 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 and mGluR6) in rod precursors located in the photoreceptor layer . 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 .
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
Expression of Retinal Progenitor Markers by Late RPCs
The GFP+ neurospheres (Fig. 1A) 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. 1B). 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 3; Fig. 2A), whereas > 86.5% expressed Nestin (Table 3; Fig. 2B), 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. 2C) and Nestin (Fig. 2D) was downregulated by adherent RPCs upon differentiation.
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.
Table 3. Quantitative evaluation of effect of CNTF on the expression of different cell markers
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. 3A). 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 3). Many of these cells adopted a bipolar morphology and extended long thin neurite-like processes, forming a network between neighboring cells (Fig. 3B).
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 and GFAP expression was determined by immunocytochemistry (Table 3; Fig. 4). After 14 days of CNTF treatment, the percentage of RPCs expressing PKC increased from 12.8% to 41.1% (Table 3). Most of the PKC-positive cells in the CNTF-treated cultures exhibited bipolar morphology (Figs. 4J–4L), whereas those in the untreated cultures remained undifferentiated (Figs. 4G–4I). CNTF also had an inductive effect on GFAP expression. The percentage of cells expressing GFAP increased significantly, from 8.2% to 48.3% (Table 3). The GFAP-positive cells in RPC cultures grown in the absence of CNTF appeared undifferentiated (Figs. 4A–4C), whereas those grown in the presence of CNTF displayed various morphologies, including cells extending many thin, neurite-like processes (Figs. 4D–4F). 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 3). In addition, RPCs grown in the absence of CNTF (Fig. 2B) displayed more intense Nestin staining compared with those grown in the presence of CNTF (Fig. 2D).
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 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 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 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. 5). RPCs grown in the absence of CNTF (referred to as RPCs in Fig. 5) 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 and GFAP. The downregulation in transcripts associated with maintaining the progenitor cell state along with a concomitant increase in PKC (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.
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) 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, 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. 6). Late RPCs grown in the absence of CNTF (referred to as RPCs in Fig. 6) expressed high levels of Nestin. In addition, a low level of PKC protein expression was also detected in these cells at baseline levels. Treatment with CNTF for 14 days resulted in a twofold increase in PKC 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. 6). 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 .
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 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 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
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
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