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发表于 2009-3-5 00:14 |只看该作者 |倒序浏览 |打印

作者：Toshihiro Inouea,b, Tetsushi Kagawab,c, Mikiko Fukushimaa, Takeshi Shimizub, Yutaka Yoshinagab, Shinji Takadad, Hidenobu Taniharaa, Tetsuya Tagab作者单位：a Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences andb Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University Graduate School of Medical Science, Kumamoto, Japan;c Division of Active Transport, National Institute fo


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      【摘要】
   Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However, only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a, a glycogen synthase kinase (GSK) 3 inhibitor, fibroblast growth factor (FGF) 2, and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67¨Cpositive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of &szlig;-catenin, and a GSK3 inhibitor, SB216763, mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2, with Ki-67¨Cpositive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells, and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.
      【关键词】 Retina Stem cell Wnt Proliferation Ciliary margin Beta-Catenin GSK inhibitor Fibroblast growth factor; h0 c7 h) e" a8 w9 L) e
   INTRODUCTION# K* i' @. O  O
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The ciliary marginal zone is widely known to contain immature retinal cells that continue to divide throughout life in both amphibians and fish . Hence, improved and efficient strategies to expand retinal stem cells are required.
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Wnt proteins are secreted lipid-modified signaling molecules that regulate cell proliferation and cell fate in various tissues in vertebrate embryos and can activate different intra-cellular signaling cascades, including the canonical pathway, c-Jun N-terminal kinase pathway, Ca2  pathway, and focal adhesion kinase pathway (reviewed by Pandur et al. .8 |  J" h" U. d8 R% F2 \1 Q; A- c
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During eye development in the chick, various components of Wnt signaling are expressed. Kubo et al.  demonstrated that Wnt2b can promote the proliferation of chick embryonic ciliary margin cells that can yield differentiated retinal progeny through activation of the canonical pathway. This finding encouraged us to explore whether Wnt signaling could promote the proliferation of adult retinal stem cells from the mammalian ciliary margin. In the present study, we demonstrate that Wnt3a increased the size of the spheres derived from ciliary margin. Wnt3a also increased the number of cells expressing Ki-67 and bromodeoxyuridine (BrdU) incorporation in triturated sphere cell cultures. More importantly, Wnt3a-treated sphere cells retained multipotency and formed a greater number of secondary spheres than nontreated cells, indicating that Wnt signaling functions on self-renewal of retinal stem cells (secondary sphere-forming ability). We further suggest that the Wnt3a-mediated increase in self-renewal involved the canonical pathway. Interestingly, the mitogenic effect of Wnt signaling was enhanced by exogenous FGF2 and attenuated by a FGF receptor inhibitor. Our results provide a basis for the development of useful strategies for the in vitro expansion of adult retinal stem cells.  g4 k" k. @# Z

MATERIALS AND METHODS6 C" i" T* L# E$ K6 v4 s

Isolation and Culture

Eight-week-old C57Bl6 mice and green fluorescent protein (GFP) transgenic mice were used to prepare adult retinal stem cells as previously reported . All cultures were maintained at 37¡ãC in 5% CO2." _2 W, F" W6 K" Q

Reverse Transcription¨CPolymerase Chain Reaction7 ~; O0 `6 z# b$ v/ {8 w# d

Total RNA was isolated from sphere cells using Trizol (Invitrogen). Reverse transcription was performed on 2 µg of RNA using Superscript II (Invitrogen) according to the manufacturer¡¯s protocol. Reverse transcription products were amplified by polymerase chain reaction (PCR) using KOD-plus (Toyobo, Osaka, Japan, http://www.toyobo.co.jp) and gene-specific primers  under standard reaction conditions with an initial 2-minute denaturation step followed by 24 to 30 cycles of 94¡ãC for 15 seconds, 58¡ãC for 30 seconds, and 68¡ãC for 60 seconds.

Immunocytochemistry
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The cells were fixed with 4% paraformaldehyde for 10 minutes and then blocked with 3% goat serum in phosphate-buffered saline containing 0.1% Triton X-100 for 30 minutes. The cells were incubated for 2 hours with one or two of the following specific primary antibodies at the stated dilutions: mouse monoclonal anti¨CKi-67 (1:200; BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com) as a cell-division marker; rat monoclonal anti-BrdU (1:40; Abcam, Cambridge, UK, http:// www.abcam.com) as an S-phase cell marker; rabbit antiactive caspase-3 (1:200; BD Biosciences) as a cell-death marker; mouse monoclonal antirhodopsin (1:200; Chemicon International, Temecula, CA, http://www.chemicon.com/) as a rod photoreceptor cell marker; mouse monoclonal antisyntaxin (1:200; Sigma-Aldrich) and rabbit polyclonal anti-Pax6 (1:500; Chemicon International) as amacrine cell markers; mouse monoclonal antiglutamine (Gln) synthetase (1:200; Chemicon International) as a M¨¹ller glia marker; and mouse monoclonal anti¨Cß-catenin (1:250; BD Biosciences). The cells were examined by epifluorescence after incubation for 30 minutes in Alexa 488- and/or Alexa 596-conjugated secondary antibodies (Molecular Probes, Eugene, OR; http://www.probes.com). Cell nuclei were counterstained with Hoechst 33258 (Nacalai Tesque, Kyoto, Japan, http://www.nacalai.co.jp). Negative controls were performed in parallel during all immunocytologic processing by omission of a primary antibody. No fluorescent labeling was observed in the negative controls. Images were obtained using an AX70 fluorescence microscope (Olympus, Tokyo, http://www.olympus-global. com) or TCS SP2 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany, http://www.leica-microsystems.com).6 b# m! W5 L& p- o2 s
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Statistical Analysis2 V$ G/ v8 L  s% z! t" o3 n/ F9 n

The data represent the mean ¡À standard deviation of three separate experiments. For cytochemical studies, five randomly selected fields per sample were analyzed in each condition. Statistical significance was determined by Student¡¯s two-tailed t-test.. Z; R- o( C  w$ P# v: d; Y

RESULTS
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Stem Cell Properties of Ciliary Margin Cells* Z$ t, q& M( {- Q: E5 e
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To ensure the existence of retinal stem cells from the ciliary margins of 8-week-old mice, the dissociated ciliary margin cells were cultured in the nonadherent condition at a clonal density. After 5 days in culture, sphere formation was observed. To further examine whether these sphere colonies were generated by the proliferation of single cells, ciliary margin cells were prepared from both GFP-expressing and wild-type mice, and the cell mixture was cultured at a clonal density for 5 days according to the method of Tropepe et al.  (Table 1). Thus, the sphere cells from the ciliary margin possessed the stem cell characters of self-renewal and multilineage potential.

Figure 1. Clonal sphere formation of retinal stem cells from the adult ciliary margin. Phase (A) and fluorescence (B) photomicrographs are shown. Dissociated adult ciliary margin cells from GFP-expressing and wild-type mice were mixed and cultured in the nonadherent condition for 5 days. The mixture of dissociated cells generated distinctive spheres. Scale bars = 100 µm.) D& ]. v2 [% J% h  _& O; W

Table 1. Sphere cells expanded by Wnt3a or SB216763 retained their multilineage potential

Effect of Wnt3a on Cell Proliferation/ q2 S1 G+ q3 W! z5 R

Because a limited number of primary spheres can be generated from individual adult eyes, improved and efficient strategies that expand the retinal stem cell pool by promoting cell proliferation are required. Wnt is a good candidate for this because it acts as a mitogen for immature retinal cells , was applied to the dissociation culture together with the recombinant Wnt3a. Sufficient dose of Fzd-8-CRD decreased the number of Ki-67¨Cpositive cells to the basal level (Fig. 2D), confirming the mitogenic effect of Wnt3a on sphere-derived cells. To examine DNA synthesis in the sphere-derived cells, they were pulse-labeled with BrdU for 4 hours. In the presence of 20 ng/ml Wnt3a, the number of BrdU-positive cells was increased from 10.2% ¡À 2.5% to 18.8% ¡À 1.6% (p % R/ V9 A2 ]% }3 M5 d8 x: ?9 V
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Figure 2. Effect of Wnt3a on the cell-division markers. (A): Expression of Fzd family and LRP-5/6 mRNAs in the primary sphere cells were examined by reverse transcription¨Cpolymerase chain reaction. Gene products specific for Fzd-1, -3, -4, -6, -7, and -8 and LRP-5 and -6 were amplified. (B¨CF): Primary spheres were triturated and cultured in the adherent condition with various concentrations of Wnt3a for 48 hours. In the control cultures, 2.5 µg/ml dimethyl sulfoxide was added to the medium. The fluorescence photomicrographs (B, C) show merged images of Hoechst (blue) and anti¨CKi-67 antibody (green) labeling. The number of Ki-67¨Cpositive cells in the culture containing 20 ng/ml Wnt3a (C) is greater than that in the control culture (B). Quantification of Ki-67¨Cpositive cells (D) shows that Ki-67¨Cpositive cells increase in a Wnt3a dose-dependent manner, and Fzd-8-CRD (a Wnt antagonist) blocks this effect. All data represent the means ¡À standard deviations of three separate experiments. The fluorescence photomicrographs (E, F) show merged images of Hoechst (blue) and anti-bromodeoxyuridine (BrdU) antibody (red). In the presence of Wnt3a, the number of BrdU-incorporating cells is greater than that in the control condition. Scale bars = 100 µm. *p
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To further estimate the proliferative response, we assessed the size of the primary spheres (50 µm or more of the diameter) with or without Wnt3a. After a 5-day culture in vitro, the diameter of the spheres was increased in the presence of 20 ng/ml Wnt3a (Fig. 3A), consistent with the data in the adherent condition described above. Notably, the average volume of the Wnt3a-treated spheres was threefold larger than that of the control spheres, although various sizes of spheres appeared in Wnt3a-containing cultures. To exclude the possibility that Wnt3a increased each cell volume, the nuclei of sphere cells were stained by Hoechst 33258 and the cell densities were examined by the confocal microscope. The cell densities of the Wnt3a-treated spheres were nearly equal to those of control spheres (Figs. 3B, 3C), confirming that the size of the spheres was reflected in the cell number but not the cell size. On the contrary, there was no significant difference between the number of primary sphere colonies in the Wnt3a-containing and control cultures (data not shown), suggesting that Wnt3a did not affect the number of cells capable of forming primary spheres nor transform nonsphere forming cells into sphere-forming stem cells. Taken together, Wnt3a promotes the proliferation of sphere-forming cells from the adult ciliary margin.

Figure 3. Effect of Wnt3a on the sphere formation. (A): Percentages of spheres categorized by their diameter. Isolated ciliary margin cells were plated at 20,000 cells/ml and cultured in the nonadherent condition with 20 ng/ml Wnt3a for 5 days, and the sphere size was estimated. In the control cultures, 2.5 µg/ml dimethyl sulfoxide (DMSO) was added to the medium. A large proportion of spheres in the Wnt3a-containing culture are greater than 100 µm in diameter, whereas most of the spheres in the DMSO-treated control culture have diameters of 100 µm or less. The results are representative of three independent experiments. The percentages of sphere-forming cells with DMSO and Wnt3a were 0.098% ¡À 0.02% and 0.12% ¡À 0.03%, respectively. (B, C): Confocal microscopic images of the nuclei-counterstained primary spheres. The cell densities of the Wnt3a-induced large-sized spheres (C) were nearly equal to that of small-sized spheres in the control condition (B). (D): Numbers of secondary spheres generated from primary spheres. Primary spheres were triturated and cultured in the nonadherent condition for 7 days in the presence of fibroblast growth factor 2 and epidermal growth factor. The number of secondary spheres generated from Wnt3a-treated spheres is greater than that from DMSO-treated control spheres. The results are the means ¡À standard deviations of three replicates. Scale bar = 100 µm. **p

Characterization of Cells Expanded by Wnt3a

Although Wnt3a promoted the proliferation of sphere cells, it remained unclear whether this proliferation was accompanied by self-renewal of the adult retinal stem cells. If Wnt3a promoted self-renewal, the Wnt3a-treated cells should retain their stem cell characters even after proliferation. To test this hypothesis, individual primary sphere colonies cultured in the presence or absence of Wnt3a were dissociated and allowed to form secondary spheres. The Wnt3a-treated cells generated fourfold the number of secondary sphere colonies compared with dimethylsulfoxide (DMSO)-treated cells from a single primary sphere (Fig. 3D). In parallel with the increased sphere size after Wnt3a treatment, the Wnt3a-treated spheres contained greater numbers of stem cells than the control DMSO-treated spheres, indicating that Wnt 3a promotes proliferation of sphere-derived cells, including retinal stem cells.. j% x, ?" C9 M! x8 y; G
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To further characterize the cells expanded by Wnt3a, we next examined the multilineage potential of Wnt3a-treated cells. Sphere colonies grown in Wnt3a-containing medium were dissociated and cultured for 7 days under conditions that promote retinal cell differentiation as described above. The Wnt3a-treated cells expressed several retinal cell¨Cspecific markers, such as rhodopsin as rod photoreceptors (Fig. 4A), Pax6 and syntaxin as amacrine cells (Fig. 4B), or Gln synthetase as M¨¹ller glia (Fig. 4C). In respect of these markers, Wnt3a-treated cells did not show any marked deviation of retinal cell fate compared with FGF2-treated cells (Table 1). Thus, sphere cells expanded by Wnt3a retained their multilineage potential.( v, j! x  j% u9 |2 e0 o# u
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Figure 4. Fluorescence photomicrographs of differentiated cells. Wnt3a-treated sphere cells were triturated and cultured under conditions that promote retinal cell differentiation for 7 days. The Wnt3a-treated cells expressed several retinal cell¨Cspecific markers, such as rhodopsin as rod photoreceptors (A), Pax6 and syntaxin as amacrine cells (B), or glutamine (gln) synthetase as M¨¹ller glia (C). Scale bars = 100 µm.- A! h$ Y+ O( D

Activation of the Canonical Wnt Pathway
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To investigate whether Wnt3a protein activates the canonical Wnt pathway in sphere-derived cells from the adult ciliary margin, we next examined the subcellular localization of ß-catenin by immunocytochemistry. ß-catenin is a coactivator of LEF/TCF-dependent transcription but is normally phosphorylated by GSK3ß and quickly degraded. Activation of the canonical Wnt pathway inhibits the kinase activity of GSK3ß, resulting in relocation of stabilized ß-catenin to the nucleus. After trituration of primary spheres derived from the adult ciliary margin, the cells were cultured in the adhesive condition for 1 day and then treated with or without 20 ng/ml Wnt3a for 2 hours. The Wnt3a-treated cells showed nuclear accumulation of ß-catenin (Fig. 5B, Wnt3a). In contrast, a low level of ß-catenin was observed under the plasma membrane and in the cytoplasmic region of nontreated cells (Fig. 5A, control). Thus, Wnt3a is capable of activating the canonical Wnt pathway in sphere-derived cells.8 D/ o7 _6 d1 m/ @

Figure 5. Nuclear translocation of ß-catenin in the sphere-derived cells induced by Wnt3a. The localization of ß-catenin was examined by immunohistochemistry at 2 hours after the addition of 2.5 µg/ml DMSO (A) or 20 ng/ml Wnt3a (B). (A): ß-catenin is observed in the cytoplasm and plasma membrane. (B): Wnt3a-treated cells show nuclear accumulation of ß-catenin. Scale bars = 100 µm.
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Effect of a GSK3 Inhibitor on Proliferation9 a1 n( m" a' T

Despite the activation of the canonical pathway by Wnt3a, it remained unclear whether its activation was involved in promoting the proliferation of sphere-derived cells. Thus, we investigated the effect of SB216763, a specific GSK3 inhibitor . SB216763 treatment of sphere-derived cell cultures increased Ki-67¨Cpositive cells in a dose-dependent manner (Fig. 6A). This effect was maximal at 2.5 µM, when the number of Ki-67¨Cpositive cells increased by twofold compared with control cultures. Moreover, the sphere size was increased by SB216763 addition (Fig. 6B). Thus, the GSK3 inhibitor alone mimicked the Wnt3a activity on the proliferation of sphere-derived cells, suggesting that activation of the canonical Wnt pathway was involved in the enhancement of proliferation of sphere-derived cells. Moreover, the SB216763-treated cells expressed several retinal cell¨Cspecific markers under conditions that promote retinal cell differentiation and did not show any marked deviation of retinal cell fate compared with FGF2-treated cells (Table 1). Thus, sphere cells expanded by SB216763 retained their multilineage potential.& y* ~* W0 k6 d& G6 z
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Figure 6. Increase in cell proliferation induced by SB216763 (a GSK3 inhibitor). (A): The percentage of Ki-67¨Cpositive cells was examined over the same time course described in the legend for Figure 2. The primary spheres were triturated and cultured in the adherent condition with SB216763 for 48 hours. Addition of 2.5 µM SB216763 increases the Ki-67-positive cells to 45% of the total cells. The results are the means ¡À standard deviations of three replicates. **p ) O/ }8 X3 w0 a6 W: n
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Cooperative Effect of FGF and Wnt Signaling  Q, k/ V) d# N, h1 h
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Adult retinal stem cells from the ciliary margin have been reported to release a small amount of endogenous FGF2 that promotes sphere formation since antibodies to FGF2 caused a reduction in the number of spheres . Addition of FGF2 at 10 ng/ml to sphere-derived cell cultures increased the Ki-67¨Cpositive cells by threefold, and this activity was reduced to the control level by 6.0 µg/ml SU5402 (Fig. 7A). SU5402 decreased Ki-67¨Cpositive cells even in the absence of exogenous FGF2, supporting that endogenous FGF2 signaling was involved in the proliferation of retinal stem cells.7 x( M1 L. i* i/ D6 p

Figure 7. Effects of fibroblast growth factor 2 (FGF2) and its inhibitor on cell proliferation. The percentage of cells expressing Ki-67 was determined as described in the legend for Figure 2. Triturated sphere cells were cultured with FGF2, SU5402 (a FGF receptor inhibitor), SB216763, or their combination for 48 hours. (A): In the presence of exogenous FGF2, SU5402 decreases the Ki-67¨Cpositive cells in a dose-dependent manner. SU5402 decreases the Ki-67¨Cpositive cells, even in the absence of exogenous FGF2, suggesting the existence of endogenous FGF in the sphere-derived cell culture. (B): The effect of SB216763 on the cell proliferation is attenuated by SU5402 and enhanced by FGF2. The results are the means ¡À standard deviations of three replicates. *p

We then added the FGFR inhibitor (SU5402) or FGF2 to the sphere-derived cell cultures in the presence of the GSK3 inhibitor (SB216763) and assessed the number of Ki-67¨Cpositive cells. Under all of the above conditions, there was no difference in the cell death estimated by immunocytochemical detection of activated caspase-3 (data not shown). Interestingly, the FGFR inhibitor attenuated the effect of the GSK3 inhibitor on cell proliferation and reduced the number of Ki-67¨Cpositive cells from 44%¨C18% (Fig. 7B). In contrast, exogenous FGF2 enhanced the proliferative effect of the GSK3 inhibitor and increased the Ki-67¨Cpositive cells to nearly 80% of the total cells, suggesting that there is an intense cooperative effect of FGF2 and Wnt signaling on cell proliferation of adult retinal sphere-derived cells.. s* F. G6 [4 \1 k3 k8 X5 P

DISCUSSION9 z5 Q& O1 g6 J" n4 w* S6 w

In the present study, we showed that Wnt3a increased the number of secondary spheres (promoting self-renewal) and that the expanded cells in the presence of Wnt3a preserved their stem cell abilities to yield differentiated progeny (maintaining multipotency), indicating that Wnt signaling has a mitogenic effect on adult retinal stem cells. It should be noted that sphere-derived cells may include retinal stem cells and committed retinal progenitor cells. Therefore, we refer to the sphere-derived cells as retinal stem/progenitor cells hereafter in this report. Wnt3a induced nuclear accumulation of ß-catenin in retinal stem/progenitor cells. More strikingly, the GSK3 inhibitor SB216763, which can activate the canonical Wnt pathway, mimicked Wnt3a activity in terms of the enhancement of retinal stem/progenitor cell proliferation. These findings indicate that the canonical Wnt pathway contributes to the proliferative effect of Wnt3a on retinal stem/progenitor cells. Thus, our study provides evidence that activation of canonical Wnt signaling is useful for expanding retinal stem/progenitor cell pools in vitro. SB216763 is a less-expensive material than recombinant Wnt3a protein and could therefore reduce the cost of tissue engineering. Although the effect of another collateral pathway cannot be excluded, there is little evidence that noncanonical Wnt pathways positively regulate the cell cycle .# g5 J- C6 R1 T/ g  D9 l

Our study further suggests that the combination of FGF2 and Wnt3a has a strong additive effect on proliferation of adult retinal stem/progenitor cells. In the absence of endogenous FGF signaling, 7% of the total cells were Ki-67¨Cpositive. In the presence of the GSK3 inhibitor but without FGF signaling (after addition of the FGF receptor inhibitor), 18% of the total cells were Ki-67¨Cpositive (Fig. 7B). If the FGF-responsive and Wnt-responsive cells represent two different populations, 25% (7% 18%) of the total cells should have been Ki-67¨Cpositive after the addition of the GSK3 inhibitor by itself. However, 44% of the cells were Ki67-positive, suggesting synergistic interaction between the FGF and Wnt signaling pathways. In other words, some of the retinal stem/progenitor cells showed cell cycle progression by activation of the canonical Wnt pathway that was dependent on endogenous FGF signaling. Concurrently with our present results, which we presented in the recent annual meeting of the Association for Research in Vision and Ophthalmology , supporting our data.

As demonstrated in this study, the adult murine ciliary margin contains Wnt signal-responsive stem cells, although they are mitotically quiescent in vivo .7 a/ g6 {9 }: R9 }

How can Wnt signaling be applied to stem cell therapy? It was recently reported that sphere cells generated from the ciliary margin could be incorporated into damaged or developing retinas, where they expressed retinal cell¨Cspecific markers, such as rhodopsin, syntaxin, and protein kinase C . Further studies are required to fully characterize the proliferation and differentiation of retinal stem cells.

CONCLUSION

Wnt3a increased the self-renewal of retinal stem cells from the adult ciliary margin via the canonical pathway. A GSK3 inhibitor could mimic the proliferative effect of Wnt3a, which was partly dependent on FGF signaling. FGF and Wnt signaling showed a synergistic effect on retinal stem/progenitor cell proliferation, stimulating more than 75% of the total cells to become Ki-67¨Cpositive proliferating cells. These results may provide a novel therapeutic strategy for in vitro pooling or in vivo activation of retinal stem cells derived from the adult ciliary margin.& W& o: l3 a& s* s$ p
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ACKNOWLEDGMENTS

The authors are very grateful to M. Ohta-Teramoto for secretarial assistance and to Y. Saiki for technical help. This work was supported by a Grant-in-Aid for 21st Century COE Research "Cell Fate Regulation Research and Education Unit" and Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology.

DISCLOSURES

The authors indicate no potential conflicts of interest.* \, x3 \( i. N
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