Differentiation of human neural progenitor cells regulated by Wnt-3a

Differentiation of human neural progenitor cells regulated by Wnt-3a

Biochemical and Biophysical Research Communications 400 (2010) 358–362 Contents lists available at ScienceDirect Biochemical and Biophysical Researc...

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Biochemical and Biophysical Research Communications 400 (2010) 358–362

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Differentiation of human neural progenitor cells regulated by Wnt-3a Rayk Hübner, Anne-Caroline Schmöle, Andrea Liedmann, Moritz J. Frech, Arndt Rolfs, Jiankai Luo ⇑ Albrecht-Kossel-Institute for Neuroregeneration (AKos), School of Medicine, University of Rostock, Gehlsheimer Strasse 20, 18147 Rostock, Germany

a r t i c l e

i n f o

Article history: Received 4 August 2010 Available online 22 August 2010 Keywords: Wnt-3a Wnt/b-catenin signaling Neural stem cells

a b s t r a c t Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and b-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized b-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/b-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized bcatenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/b-catenin pathway in ReNcell VM cells. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction In recent years, neural stem and progenitor cells (referred to as neural precursor cells – NPCs) have been suggested to be a promising source for transplantation strategies to replace neurons lost in neurological diseases, such as neurodegenerative diseases, stroke or spinal cord injury [1]. Although significant progress has been made for application of NPCs in the clinics, key questions about neuronal and glial cell fate determination remain to be answered [2]. Understanding the regulation of differentiation is essential for controlling NPC differentiation in stem cell engineering. The efficacy of cell based therapies may depend on the ability to control the proliferation, differentiation and survival of NPCs, and as such much stem cell research is focused on these areas [3]. Wnt/b-catenin signaling has been shown to be involved in controlling differentiation processes of NPCs [4,5]. However, little is known about the influence of Wnt/b-catenin signaling on differentiation in human NPCs. Recently, inhibition of GSK-3b using small molecules has been shown to enhance neuronal differentiation in human fetal mesencephalic NPC-line ReNcell VM [6]. Here, we have investigated the impact of the Wnt/b-catenin signaling pathway on the neuronal and glial differentiation in ReNcell Abbreviations: bFGF, basic fibroblast growth factor; Dkk-1, Dickkopf-1; EGF, epidermal growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; G6PD, glucose-6-phosphate-dehydrogenase; GSK3, glycogen synthase kinase-3; HAS, human serum albumin; LEF1, lymphoid enhancer-binding factor; LRP6, low density lipoprotein receptor related protein 6; NPC, neural progenitor cell; TCF, Tcell factor. ⇑ Corresponding author. Fax: +49 381 4944899. E-mail address: [email protected] (J. Luo). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.066

VM NPCs. The ReNcell VM cells can differentiate into neurons, astrocytes and oligodendrocytes within a few days of differentiation in vitro [6–8], which favors its use as a model system to analyze differentiation mechanisms. Our results show that activation of the Wnt/b-catenin pathway by transfection of mutant-stabilized b-catenin or Wnt-3a protein induces Wnt target gene expression and activation of a TCF-reporter. However, in contrast to mutant-stabilized b-catenin, Wnt-3a can promote neuronal differentiation but not gliogenesis. Furthermore, the treatment with Dkk-1 or transfection with Axin did not alter neuro- or gliogenesis. Thus, our results suggest an enhancement of neurogenesis by Wnt-3a in human NPCs by a mechanism independent of the transcriptional activity of b-catenin. 2. Materials and methods 2.1. Reagents and cell line Recombinant mouse Wnt-3a and human Dkk-1 proteins were purchased from R&D (Wiesbaden, Germany). For preparation of stock solutions, proteins were dissolved in phosphate buffered saline (PBS) containing 0.1% human serum albumin (HSA). The ReNcell VM cell line (Millipore, Billerica, USA) used in this study is derived from ventral mesencephalic tissue of a 10-week-old human fetus and is immortalized by retroviral transduction with vMyc (ReNeuron Ltd., Guildford, UK) [8]. 2.2. Cell culture ReNcell VM cells were cultivated essentially according to the protocol described previously [6].

R. Hübner et al. / Biochemical and Biophysical Research Communications 400 (2010) 358–362

2.3. Plasmid construction A mouse mutant S33Y b-catenin sequence with a C-terminal HA-tag was amplified using primers of 50 -ctcgaggaaccaccatggctactcaag-30 and 50 -ctcgagttaagcgtaatctggaacatcatatgggtacaggtcagtatcaaaccaggc-30 by RT-PCR using pCGN-S33Y [9] as a template. Sequence of C-terminally HA-tagged murine Wnt-3a was amplified from pUSEamp-Wnt-3a vector (Upstate Biotechnology) using primers of 50 -ctcgagtcaccaccatggctcctc-30 and 50 -ccagggcattggttaagcgtaatctg-30 . Sequence of human Axin was amplified from ReNcell VM cDNA using primers of 50 -ctcgaggaaccaccatggatatccaag-30 and 50 -ctcgagtcagtctaccttctccactttgc-30 . Real-time RT-PCR parameters were used as following: initial denaturation at 95 °C for 5 min, then 30 s at 95 °C for denaturation, 30 s at 62 °C for annealing and 2 min at 72 °C for extension with 40 cycles. The resulting fragments (S33Y, 2.3 kb; Wnt-3a, 1.1 kb and Axin, 2.5 kb, respectively) were cloned into pGEM-T easy vector (Promega) and identified by sequencing. Fragments were further cut out using XhoI and subcloned into XhoI site of pCAGGS [10]. 2.4. Luciferase reporter gene assay TCF-dependent transcription was analyzed by the SuperTOPFlash reporter gene assay [11]. Cells (1  106) were transfected with 2 lg of SuperTOPFlash and 4 ng of pRL-TK (Promega, Mannheim, Germany) using Nucleofector technology (Lonza, Cologne, Germany). Twenty-four hours after transfection the cells were harvested for luciferase assay using the Dual Luciferase Assay Kit (Promega). The activity of the firefly luciferase and constitutively expressed Renilla luciferase, which was used to normalize for transfection efficiency, were measured in triplicate using the luminometer LB 9508 (Berthold, Bad Wildbad, Germany). 2.5. Quantitative real-time RT-PCR To quantify mRNA in cells, a LightCycler (Roche, Mannheim, Germany) in combination with the LightCycler 3.52 software was used. The first-strand cDNA was synthesized using FastLane cDNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. PCR was performed using the FastStart DNA SYBRGreen Plus Kit (Roche) according to manufacturer’s instructions. PCR products were verified by size in agarose-gel electrophoresis, sequencing and melting point analysis. Relative changes of mRNA amount were calculated using the DDCT method [12]. Tested gene mRNA amount was normalized to the housekeeping gene glucose6-phosphate-dehydrogenase (G6PD). All samples were run in duplicate. Cycling parameters were as follows: initial denaturation at 95 °C for 12 min; 45 cycles of 1 s at 95 °C for denaturation, 5 s at 62 °C for annealing and 10 s at 72 °C for extension. Primer sequences were used as following: Axin2: 50 -agtcagcagagggacag gaa-30 and 50 -agctctgagccttcagcatc-30 ; G6PD: 50 -atcgaccactacctggg caa-30 and 50 -ttctgcatcacgtcccgga-30 [13]; LEF1: 50 -gacgagatgatccc cttcaa-30 and 50 - agggctcctgagaggtttgt-30 ; cyclinD1: 50 -acctggatgctg gaggtct-30 and 50 -gctccatttgcagcagctc-30 [14].


goat anti-mouse Alexa Fluor 680, and goat anti-mouse IRDye 800 (all from Invitrogen, 1:10,000). 2.7. Flow cytometry Flow cytometry analysis was performed as described previously [6]. Briefly, for each condition, 50,000 cells were counted and analysis was done using FACSCalibur (BectonDickinson) in combination with CellQuest Pro software. Aggregated cells and debris revealed by forward scattering were filtered out of the data set prior to analysis. Gates were set using cells stained with negative control antibodies. Primary antibodies used were as follows: mouse anti-HuC/D (Invitrogene; 1:100); mouse anti-b-tubulin III (Tuj1) (sc-51670; Santa Cruz; 1:100); rabbit anti-GFAP (Dako; 1:500). Secondary antibodies used were as follows: goat antirabbit AlexaFluor488; goat anti-mouse AlexaFluor 488; goat antirabbit AlexaFluor 647 (all Invitrogen; 1:1000). 2.8. Statistical analysis Results are reported as means ± SEM from at least three independent experiments. Statistical evaluation was carried out using the two-tailed Student’s t-test with Excel software (Microsoft, USA). Difference was considered to be significant when pvalue < 0.05. 3. Results and discussion To test whether the cells are able to respond to Wnt signaling, proliferating cells were treated for 4 h with recombinant Wnt-3a (50 ng/ml), an activator of canonical Wnt signaling in a variety of cell systems [15,16] and lithium chloride (LiCl; 20 mM), which is widely used to activate canonical Wnt signaling [17,18]. Treatment with Wnt-3a resulted in an increase of the phosphorylated Wnt coreceptor LRP6 (LDL-receptor-related protein) at Ser1490 position (Fig. 1B). Phosphorylation of LRP6 could be confirmed by detecting a shift of total LRP6 due to hyperphosphorylation (Fig. 1A). Phosphorylation of LRP6 is essential for b-catenin dependent transcription [19]. Furthermore, the mediator proteins Dishevelled (Dvl) 2

2.6. Western blotting The Western blot analysis procedure has been described previously [7]. The following primary antibodies were used: mouse anti-HA (6E2; Cell Signaling; 1:10,000); mouse anti-b-catenin (E5; Santa Cruz; 1:2000); rabbit anti-Axin (34-5900; Zymed; 1:1000); mouse anti-GAPDH (6C5, Abcam, 1:10,000); mouse antiactin (AC-15; Sigma, 1:10,000); rabbit anti-Dvl2 (#3216; Cell Signaling; 1:1,000); mouse anti-Dvl3 (4D3; Santa Cruz; 1:500); rabbit anti-LRP6 (C5C7; Cell Signaling; 1:2000). The following secondary antibodies were used: goat anti-rabbit Alexa Fluor 680,

Fig. 1. Activation of Wnt signal transducers and b-catenin under proliferating conditions shown by Western blotting. Proliferating ReNcell VM cells were stimulated for 4 h with Wnt-3a (50 ng/ml), LiCl (20 mM) or an equivalent amount of 0.1% HSA in PBS as control. Total and cytoplasmatic lysates were prepared and analyzed by Western blot using antibodies against indicated proteins. Hyperphosphorylated protein bands are marked with black arrows. White arrows indicate non-phosphorylated protein form. GAPDH was used as a loading control.


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and Dvl3 were also activated, as indicated by a shift of the mature form (white arrowhead) to the phosphorylated from (black arrowhead) upon Wnt-3a stimulation (Fig. 1C and D). Moreover, treatment with Wnt-3a increased cytosolic b-catenin level in cells (Fig. 1E). As expected, treatment with LiCl did not alter the phosphorylation status of LRP6, Dvl2 or Dvl3, but increased the cytosolic b-catenin by inhibition of glycogen synthase kinase-3b (GSK-3b) activity [17,18]. These results show that at the protein level the ReNcell VM cells express the essential co-receptor LRP6, as well as the mediators of canonical and noncanonical Wnt signaling Dvl2 and Dvl3. The treatment with Wnt-3a and LiCl resulted in activation of b-catenin, clearly showing that Wnt/b-catenin signaling is inducible in these cells. To our knowledge, this is the first study showing activation of LRP6 and Dvl proteins upon Wnt pathway stimulation in human NPCs. To confirm the expression of generated plasmids, cells were transfected, and 48 h after transfection (time point 0 h for differentiation), growth factors were removed to induce differentiation, subsequently cells were cultivated for up to 3 days. Samples from each time point were taken and analyzed by Western blotting and results show that Wnt-3a, stabilized b-catenin and Axin proteins could be detected at each time point (Supplementary Fig. S1). Next we asked whether Wnt-3a and mutant-stabilized b-catenin are able to upregulate T-cell factor (TCF)-mediated transcription using pSuper12TopFlash plasmid, which drives the expression of luciferase via a promoter containing 12 TCF-binding sites. When Wnt-3a or mutant-stabilized b-catenin was co-transfected together with pSuper12TopFlash plasmid, the TCF-activity is increased 6-fold upon Wnt-3a cotransfection and 12-fold upon cotransfection with mutant-stabilized b-catenin compared to control (set as 1-fold; Fig. 2) at 24 h after transfection. These results clearly show that ReNcell VM cells possess the cellular machinery needed to activate TCF-mediated transcription. Furthermore, to investigate the Wnt target genes in NPCs, such as Axin2, LEF1 and Cyclin D1 [20], the cells were transfected with GFP control vector alone, Wnt-3a and stabilized-mutant b-catenin, respectively, and transcription levels of Wnt/b-catenin target gene were measured by quantitative real-time PCR at 24 and 48 h after transfection in proliferation medium (Fig. 3). Wnt-3a overexpression resulted in an increase of Axin2 expression about 13-fold at 24 h and about 5.7-fold at 48 h compared to the control, while stabilized b-catenin increased Axin2 about 6-fold at 24 h and 1.6-fold at 48 h (Fig. 3A). LEF1 expression was increased by 5-fold after 24 h and 8-fold after 48 h of Wnt-3a overexpression, and by about 5-fold after 24 and 48 h of mutant-stabilized b-catenin (Fig. 3B). Cyclin D1 expression was only induced by Wnt-3a and mutantstabilized b-catenin after 48 h (both about 5-fold; Fig. 3C). Thus, Fig. 3. Inducibility of Wnt target genes in ReNcell VM cells. Cells were transfected with indicated vectors and after 24 or 48 h in proliferation medium, samples were taken and mRNA levels of the target genes Axin2 (A), LEF1 (B) and cyclin D1 (C) were detected by quantitative real-time RT-PCR. Values are normalized to G6PD (glucose-6-phophate-dehydrogenase). All Data are normalized to GFP vector transfected cells (time point control, indicated as black line) and presented as means ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control.

Fig. 2. Inducibility of TCF-activity in ReNcell VM cells. Cells were transfected with TOP and pCAGGS plasmids (indicated) and TCF-activity was measured after 24 and 48 h in proliferation medium. Data are normalized to control (pCAGGS-GFP transfected cells, indicated as black line) and presented as means ± SEM from at least three independent experiments. ***p < 0.001 compared to the control.

target genes known to be induced by Wnt/b-catenin signaling in murine systems [20] are also inducible in human NPCs. Stimulation of canonical Wnt signaling of NPCs from the middle, neurogenic phase, but not from the early and expansion phases, has been shown to increase neuronal differentiation [4,5,21], demonstrating a distinct response to Wnt signals by cells in different developmental stages. Therefore, we analyzed whether differentiation of ReNcell VM cells was altered by transfection of Wnt-3a or stabilized b-catenin. After the cells were transfected with plasmid and grown in proliferation medium for 48 h, the culture medium was changed for a differentiation medium without

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growth factors (time point 0 h for differentiation). Up to 3 days after initiation of differentiation, samples were collected and the mRNA levels of the neuronal markers HuC and Tuj1 were analyzed. Overexpression of Wnt-3a induced an increase of HuC expression by about 1.5-fold and of Tuj1 by about 1.2-fold compared to control at time points 0 h, 2 d and 3 d (Fig. 4A and B), whereas overexpression of stabilized b-catenin had no effect (Fig. 4A and B). These data suggest that Wnt-3a is able to partially overcome the effects of growth factors to induce expression of neuronal markers. However, cells transfected with Wnt-3a did reveal a phenotype comparable to control cells under proliferating conditions (data not shown). To analyze whether the increase of mRNA levels of HuC and Tuj1 results in an increase of the cell number positive for HuC/D and Tuj1, cells were transfected with Wnt-3a, stabilized b-catenin


or the Wnt inhibitor Axin [5,22] and the number of cells positive for HuC/D and Tuj1 expression was measured by flow cytometry 3 days after differentiation. Results showed that activation of Wnt/b-catenin signaling by overexpression of mutant-stabilized b-catenin or inhibition of Wnt/b-catenin signaling by overexpression of Axin had no significant effect on the percentages of HuC/ D- and Tuj1-positive cells compared to control (Fig. 4C and D). In contrast, overexpression of Wnt-3a increases HuC/D-positive cells from 5.3% (control) to 8% (p = 0.06) (Fig. 4C) and Tuj1-positive cells from 3% (control) to 4.6% (p = 0.06) (Fig. 4D). In all cases, the percentages of GFAP-positive cells had not changed compared to the control (data not shown), suggesting that they have no effect on glial differentiation. Moreover, we also treated the cells with recombinant Wnt-3a (100 ng/ml) and Dkk-1 (50 ng/ml), a potent Wnt antagonist [18], respectively. Three days after differentiation,

Fig. 4. Influence of Wnt-3a and Wnt signaling modulators on expression of neuronal and glial markers in ReNcell VM cells. (A, B) Cells were transfected with indicated vectors and after 48 h in proliferation medium (time point 0 h), cells were initiated for differentiation by removing growth factors. After 2 and 3 days, samples were collected and mRNA levels of HuC (A) and Tuj1 (B) were detected by quantitative real-time RT-PCR. Values are normalized to G6PD of control pCAGGS-GFP transfected cells (indicated as a black line) and presented as means ± SEM from at least four independent experiments. (C, D) Cells were transfected with indicated vectors and cultured in proliferation medium for 48 h. Then differentiation was initiated by withdrawl of growth factors (time point 0 h) and cells cultured for 3 days were measured by flow cytometry analysis for the markers HuC/D (C) and Tuj1 (D). (E, F) Differentiation was initiated by withdrawling growth factors and adding Wnt-3a (100 ng/ml), Dkk-1 (50 ng/ml) or an equivalent volume of 0.1% HSA in PBS (control). Cells were cultured for 3 days before being processed for flow cytometry analysis for the markers HuC/D (E) and Tuj1 (F). Data are presented as means ± SEM, from at least three independent experiments. *p < 0.05 compared to control.


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the number of HuC/D-positive cells increases from 5.8% to 8% and Tuj1-positive cells from 3.4% to 4.5% upon the stimulation of Wnt3a (Fig. 4E and F), whereas Dkk-1 had no effect on the expression of HuC/D and Tuj1 (Fig. 4E and F). In contrast, Wnt-3a stimulation did not significantly alter the percentages of cells positive for glial marker GFAP (data not shown), which is in accordance with Wnt-3a overexpression results. Interestingly, we found that activation of Wnt/b-catenin by overexpression of stabilized b-catenin or inhibition of Wnt/b-catenin by overexpression of Axin or treatment with Dkk-1 neither increase the mRNA levels of HuC/D and Tuj1 of cells, nor affect the percentages of the cells expressing neuronal markers of HuC/D and Tuj1 (Fig. 4). In contrast, Wnt-3a increases both mRNA levels and the cell percentages of HuC/D and Tuj1 (Fig. 4), suggesting that Wnt-3a is able to regulate neurogenesis in ReNcell VM cells independently of b-catenin mediated transcription. However, since Wnt-3a and stabilized b-catenin did induce Cyclin D1 expression in a similar extend and GSK3 inhibition did inhibit proliferation in this cell system [6], a mechanism involving increased proliferation of committed neuronal precursors [23] seems to be unlikely. Interestingly, inhibition of GSK-3b using small molecules also increased neurogenesis in this cell line [6], pointing out to a potential role of GSK inhibition, but not b-catenin-mediated transcription in this process. These data are in contrast to another study in human NPCs showing that Wnt-3 reduces neuronal differentiation but promoted glial differentiation, whereas, inhibition of Wnt signaling by Axin overexpression did improve neuronal differentiation [22]. The reason for this phenomenon may be differences in the nature of these cells and the stage-specific effects of Wnt signaling in NPCs. Further studies should be performed to investigate the underlying molecular mechanism of neurogenesis driven by Wnt-3a in ReNcell VM cells. In summary, our data suggest that Wnt-3a increases neuronal differentiation without affecting glial differentiation in ReNcell VM cells by a mechanism independent of the transcriptional activity of Wnt/b-catenin pathway. These results broaden the knowledge of molecular mechanisms involved in the neurogenesis of human NPCs, providing potential targets for directing differentiation of human NPCs for clinical applications. Acknowledgments RH was supported by the German Research Foundation of GRK 1387 dIEM oSiRiS. We thank Dr. R.T. Moon for the gift of the SuperTOPFlash vector, Dr. A. Ben-Ze’ev for pCGN-S33Y plasmid, Dr. H. Ogawa for pCAGGS-GFP plasmid, and E. Ewald and N. Krüger for excellent technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.08.066.

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