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Keywords:

  • Embryonic stem cells;
  • Wnt pathway;
  • Dopaminergic neurons;
  • Wnt/β-catenin signaling

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Embryonic stem cells (ESCs) represent not only a promising source of cells for cell replacement therapy, but also a tool to study the molecular mechanisms underlying cellular signaling and dopaminergic (DA) neuron development. One of the main regulators of DA neuron development is Wnt signaling. Here we used mouse ESCs (mESCs) lacking Wnt1 or the low-density lipoprotein receptor-related protein 6 (LRP6) to decipher the action of Wnt/β-catenin signaling on DA neuron development in mESCs. We provide evidence that the absence of LRP6 abrogates responsiveness of mESCs to Wnt ligand stimulation. Using two differentiation protocols, we show that the loss of Wnt1 or LRP6 increases neuroectodermal differentiation and the number of mESC-derived DA neurons. These effects were similar to those observed following treatment of mESCs with the Wnt/β-catenin pathway inhibitor Dickkopf1 (Dkk1). Combined, our results show that decreases in Wnt/β-catenin signaling enhance neuronal and DA differentiation of mESCs. These findings suggest that: 1) Wnt1 or LRP6 are not strictly required for the DA differentiation of mESCs in vitro, 2) the levels of morphogens and their activity in ESC cultures need to be optimized to improve DA differentiation, and 3) by enhancing the differentiation and number of ESC-derived DA neurons with Dkk1, the application of ESCs for cell replacement therapy in Parkinson's disease may be improved. STEM CELLS 2009;27:2917–2927


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Embryonic stem cells (ESCs) possess several unique features that make them ideal tools for studying development and for advancing therapies for devastating diseases. ESCs do not senesce, require no immortalization for long-term cultivation, and have the unique ability to self-renew and differentiate into all cell lineages [1]. These properties make ESCs an unlimited cell source for cell replacement therapy (CRT) in degenerative diseases. One such disorder is Parkinson's disease, where midbrain dopaminergic (DA) neurons from substantia nigra progressively degenerate, resulting in reduced movements, rigidity, and tremor. CRT, by replacing DA neurons, offers the only current long-term prospect for treatment of the disease. In order to safely and efficiently differentiate ESCs into DA neurons, a detailed understanding of the molecular mechanisms and signaling pathways involved in the development and generation of these midbrain DA neurons is required. In this regard, Wnt signaling, a pathway involved in the regulation of many aspects of neural development, including neural induction, proliferation, fate determination, cell migration, neurulation, and axonal growth [2–4], has also been found to regulate various stages of ventral midbrain (VM) DA neuron development [1, 5]. Wnt ligands act as morphogens and activate several downstream pathways. Perhaps the best defined is the Wnt/β-catenin pathway, also referred to as the canonical Wnt signaling pathway [6]. Wnt1, the prototypical ligand of Wnt/β-catenin signaling pathway, has been described as important for several aspects of midbrain and DA neuron development, including proliferation of progenitors in vitro [7, 8] and in vivo [9–11], VM neurogenesis [7, 12], specification of DA progenitors, and survival of DA neurons [13]. Other branches of Wnt signaling are collectively referred to as noncanonical, and they comprise a growing list of pathways, including the Wnt/planar cell polarity (PCP) and Wnt/Ca2+ pathways as two of the most intensively studied [14–16]. Although no function in DA neuron development has been attributed to the Wnt/Ca2+ pathway, the Wnt/PCP pathway has been described to play a role in VM morphogenesis in vivo [17] and in DA differentiation in vitro and in vivo [7, 17]. Interestingly, fine tuning of Wnt signaling is enabled by several soluble inhibitors such as Dickkopfs (Dkks) and secreted frizzled-related proteins (SFRPs), that interfere with extracellular components of Wnt signaling machinery [18]. However, the function of Dkks and SFRPs in DA neuron development is not known. Moreover, the role of Wnt/β-catenin signaling in the DA differentiation of ESCs has not yet been fully addressed. To address these questions, we used a panel of mouse ESC (mESC) lines lacking either Wnt1 or one of its receptors, the low-density-lipoprotein receptor-related protein 6 (LRP6). Additionally, wild-type mESCs were treated with the Wnt/β-catenin pathway inhibitor, Dkk1. Thus, by blocking canonical Wnt signaling in three different ways, we provide evidence that inhibition of this pathway promotes neuroectodermal and DA differentiation in mESC cultures leading to significant increases in the yield of mESC-derived DA neurons.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Cell Culture, Growth Factors, and Reagents

First, mESCs deficient for Wnt1 or LRP6 (Wnt1 ko and LRP6 ko) as well as their wild-type counterparts (Wnt1 wt and LRP6 wt) were propagated in undifferentiated states on feeder layers of mitomycin-treated mouse embryonic fibroblasts (MEFs) as described previously [19]. Details of the derivation of Wnt1 and LRP6 mESCs are described here [19, 20]. For the purpose of removing MEFs from the expanding mESC cultures, mESCs were trypsinized for 5 minutes in 0.05% trypsin-EDTA and plated on 6-cm culture dish coated with gelatin. After 30 minutes, most of the MEFs were attached to the plate surface. Nonattached mESCs and the few remaining MEFs were replated with media on another gelatin-coated culture dish. This led to efficient elimination of MEFs. R1 mESCs were cultured as feeder free as described elsewhere [21]. Differentiation into DA neurons was performed using the modified protocols from Barberi et al. [22] (coculture with stromal cell line PA6, subsequently referred to as the PA6-based protocol) and Ying et al. [23] (feeder-free differentiation). Briefly, for the PA6-based protocol, mESC were seeded at a density of 15-25 cells/cm2 on mitomycin-treated stromal cells PA6 and cultured in ES-SRM (KnockOut-DMEM, 15% KnockOut serum replacement, 0.1 mM β-mercaptoethanol, glutamine, 1% nonessential amino acids, 10,000 U/ml penicillin/streptomycin). After 5 days, medium was changed and supplemented with 250 ng/ml sonic hedgehog (Shh) and 25 ng/ml fibroblast growth factor 8 (FGF8). From day 8 to day 12, cells were cultured in N2 medium consisting of F12 and MEM mixture 1:1, N2 supplement, 15 mM HEPES, glutamine, 3 mg/ml albumax) supplemented with 250 ng/ml Shh, 25 ng/ml FGF8 and 20 ng/ml basic FGF (bFGF). From days 12-14, the media was replaced with N2 medium supplemented with 30 ng/ml brain derived neurotrophic factor (BDNF), 30 ng/ml glial derived neurotrophic factor (GDNF), and 200 μM ascorbic acid. For differentiation using the feeder-free protocol, mESC were seeded out (10,000-15,000 cells/cm2) on gelatin-coated 24-well plate in ES-SRM. After 24-48 hours, the media was changed to N2/B27 supplemented with or without 10-20 ng/ml bFGF, 25 ng/ml FGF8, and 250 ng/ml Shh. At day 5 and 8 of differentiation, medium was changed to new N2/B27 (no Shh, bFGF, and FGF8 added). For experiments involving the antagonism of the Wnt pathway, Dkk1 (100 ng/ml) was added to N2/B27 media at day 1 of differentiation. Controls were treated with vehicle (0.1% bovine serum albumin in phosphate buffered solution) Media was changed at day 5 and 8 of differentiation (supplemented with Dkk1 in case of treatment condition). All cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen. com), all recombinant factors were from R&D Systems Inc. (Minneapolis, MN, http://www.rndsystems.com).

Immunohistochemistry and Cell Counting

Following in vitro differentiation, the cells were fixed in 4% paraformaldehyde (+4°C for 30 minutes) and serum blocked and incubated in the appropriate primary and subsequently secondary antibodies as described previously [24]. Nuclear counterstaining was performed using Hoechst. The following antibodies were used: mouse monoclonal anti-βIII-tubulin (TuJ1; 1:1,000, Promega, Madison, WI, http://www.promega.com), rabbit polyclonal anti-tyrosine hydroxylase (TH; 1:200, Pel-Freez, Rogers, AK, http://www.invitrogen.com), Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 donkey anti-rabbit (both 1:1,000, Molecular Probes Eugene, OR, http://probes.invitrogen.com, and Invitrogen). The number of immunoreactive colonies from the total number of colonies was counted from three wells per experimental condition for each experiment. All experiments were repeated on at least three independent occasions.

Reverse Transcription Polymerase Chain Reaction and Quantitative Polymerase Chain Reaction

Samples from the PA6-based differentiation were prepared by manually isolating colonies from PA6 feeder layer under magnification. Total RNA was extracted using RNeasy extraction kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer's instructions, and reverse transcription and quantitative polymerase chain reaction (PCR) was performed as described previously [7]. As an internal control, 18S was used. Relative mRNA expression was calculated as a fold-change versus control. Primers and conditions for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) were as follows: Wnt1 (GAT TGC GAA CGC TGT TTC and TCC TCC ACG AAC CTG TTG ACG G, 56°C, 35x), Pitx3 (GGA ATC GCT ACC CTG ACA TGA G and TGA AGG CGA ACG GGA AGG TCT, 62°C, 36x), Lmx1a (GAG ACC ACC TGC TTC TAC CG and CCT CCT TCA GGA CAA ACT CG, 60°C, 35x), and Foxa2 (CAT CCG ACT GGA GCA GCT A and CAT AGG ATG ACA TGT TCA TGG AG, 60°C, 35x). All remaining primers and PCR conditions were according to those previously described [7].

Luciferase Reporter Assay and Cell Transfection

Next, mESCs seeded on gelatin-coated 24-well plate (15,000 cells/cm2) 24-30 hours were transfected with 500 ng SuperTop-Flash/SuperFOP-Flash and 50 ng of Renilla-expressing vector pRL-TK Luc (Promega, Madison, WI, http://www.promega.com) using Superfect (Qiagen) according to the manufacturer's instructions. Twelve hours after transfection, cells were stimulated with recombinant Wnt3a (100 ng/ml, R&D systems) for 22 to 24 hours. Reporter activity was measured using dual luciferase reporter assay (Promega). Analyzes of each experimental condition was performed in triplicates. Background luciferase activity (mESCs transfected only with pcDNA plasmid) was subtracted from each analyzed sample. Luciferase activity of SuperTOP/FOP-Flash was normalized to Renilla luciferase signal.

Western Blot

Differentiated colonies of mESCs were manually isolated from PA6 feeder layer under magnification. Sample processing and Western blot (WB) analysis was performed as previously described [25]. The following primary antibodies were used: mouse monoclonal anti-beta-actin (1:5000, Abcam), mouse monoclonal anti-TuJ1 (1:5,000, Promega), rabbit polyclonal anti-TH (1:1,000, Pel-Freeze), rabbit polyclonal anti-Phospho-LRP5/6 (1:1,000, Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), mouse monoclonal anti-β-catenin (1:5,000, BD Transduction Laboratories, Lexington, KY, http://www. bdbiosciences.com), mouse monoclonal anti-Dishevelled 3 (Dvl3, 1:500), rabbit polyclonal anti-Oct3/4 (1:500, both from Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) mouse monoclonal anti-active β-catenin (ABC; 1:1,000, Upstate, Charlottesville, VA, http://www.upstate.com). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies A7282 and A6667, respectively, were purchased from Sigma, St. Louis, http://www.sigmaaldrich.com.

Statistical Analyses

Statistical analyses (Student's t test, one-way analysis of variance (ANOVA)) were performed using Prism4 (GraphPad Software, Inc. La Jolla, CA, http://www.graphpad.com). p < .05 was considered a statistically significant difference (*), p < .01 (**), p <.001 (***). Results are presented as mean ± SEM.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

To analyze the role of Wnt signaling in the differentiation of ESCs into DA neurons, we used mESC lines lacking Wnt signaling components such as the Wnt1 ligand or the LRP6 receptor [19]. The genotype of each mESC line was confirmed by PCR (Supporting Fig. 1A). When examined in their undifferentiated state, knockout (ko) cell lines and their corresponding wild-type (wt) control littermate cell lines displayed no obvious differences in colony morphology. Similarly, no differences were found in the expression of pluripotency markers such as Oct4 or alkaline phosphatase (Supporting Fig. 1B, 1C, and data not shown).

Deletion of Wnt1 Increases Neuronal Differentiation and the Number of Dopaminergic Neurons in mESC Cultures

To differentiate mESC into DA neurons with ventral midbrain characteristics we used a well established protocol combining coculture of mESCs with the stromal cell line PA6 [26] and treatment with soluble factors Shh, FGF8, and FGF2 [22]. Cells were seeded (25 cells/cm2) on mitotically-inactivated PA6 and allowed to differentiate for 14 days (see Materials and Methods for details). At the final stage of differentiation (day 14), the proportion of βIII-tubulin (TuJ1; a neuronal marker) positive colonies was significantly higher in Wnt1 ko cells compared with Wnt1 wt cells (mean ± SEM: 86.33% ± 3.63, and 54.17% ± 4.64 respectively, p = .0046**, n = 6). Interestingly, almost all cells within the Wnt1 ko colonies expressed TuJ1 (Fig. 1A, 1B). Surprisingly, although Wnt1 ko mice show a near complete loss of midbrain DA neurons in vivo [13, 27], colonies positive for tyrosine hydroxylase (TH; the rate limiting enzyme in dopamine synthesis and a marker of dopaminergic neurons) were more abundant in Wnt1 ko (57.33% ± 2.80) compared with Wnt1 wt cells (33.50% ± 4.05, p = .0075**, n = 6, Fig. 1B). TH+ neurons derived from Wnt1 wt and Wnt1 ko cells showed typical neuronal morphologies (Fig. 1C). Furthermore, the number of TH+ cells per μm2 within TH+ colonies was also significantly higher in Wnt1 ko colonies (0.052 ± 0.0084) than in Wnt1 wt (0.017 ± 0.004, p = .0025**, n = 6, Fig. 1C, 1D).

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Figure 1. Absence of Wnt1 promotes mouse embryonic stem cell (mESC) differentiation into TH+/TuJ1+ cells with characteristics of ventral midbrain dopaminergic neurons, using a PA6-based protocol. (A): Wnt1 wild-type (wt) and knockout (ko) colonies immunostained for TH (red) and TuJ1 (green) after 14 days of differentiation. (B): Quantification of the percentage of TH and of TuJ1 positive colonies, showing an increase in Wnt1 ko compared with Wnt1 wt conditions after 14 days of differentiation (p < .01, n = 6). (C): High magnification of Wnt1 wt and mESCs following 14 days of differentiation immunostained for TH (red) and TuJ1 (green), with Hoechst nuclear counterstaining (blue). (D): Quantification of the number of TH+ cells per μm2 of the TH+ colonies obtained after differentiation of Wnt1 wt and ko mESCs (p < .01, n = 6). (E): Western blot analyses showing increased expression of TH and TuJ1 in Wnt1 ko condition after 10 days of differentiation. (F): Reverse transcription-polymerase chain reaction showing expression of early and late dopaminergic markers (Foxa2, Lmx1a, Nurr1, Pitx3, TH, and dopamine transporter) in differentiated wt and ko cells. PA6 cells were used as negative control. (G): Relative mRNA expression of Nurr1 (p = .0499) and TH (p = .0548). Graphs present gene expression as a fold change of transcript level in Wnt1 ko compared with Wnt1 wt (n = 3). Abbreviations: DAT, dopamine transporter; ko, knockout; TH, tyrosine hydroxylase; wt, wild-type. TuJ1, bIII-tubulin; Foxa2, forkhead box transcription factor A2; Lmx1a, LIM homeobox transcription factor 1a; Nurr1, nuclear receptor related transcription factor 1; Pitx3, Paired-like homeodomain transcriptionfactor 3.

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Our observation that the absence of Wnt1 enhances the number of TuJ1 and TH-positive cells was confirmed by western blot analyses illustrating that, at a protein level, both TuJ1 and TH increased in Wnt1 ko cells after both 10 and 14 days of differentiation (Fig. 1E and data not shown). We also confirmed by RT-PCR that the neurons obtained in our culture conditions expressed typical markers of ventral midbrain DA neurons such as: TH, Nurr1 (a marker for DA neurons, and DA precursors), Paired-like homeodomain transcription factor 3 (Pitx3; specific for midbrain DA neurons and critical for their survival), dopamine transporter (DAT; a marker expressed in mature DA neurons), as well as Lmx1a (a homeodomain transcription factor important for DA neuron fate specification [28]) and Foxa2 (a forkhead transcription factor important for DA neuron specification and survival [29]) (Fig. 1F). Moreover, when the levels of Nurr1 and TH mRNA were examined by quantitative PCR, increases were detected in differentiated Wnt1 ko cells compared with wt cells (Fig. 1G), confirming the increase in DA neuron yield.

Increased Dopaminergic Differentiation in Lipoprotein Receptor-Related Protein 6 Knockout mESCs

Subsequently we induced the differentiation of LRP6 mESCs, employing the same protocol as for Wnt1 cells. Following 14 days of differentiation, most of both the LRP6 wt and the LRP6 ko colonies were immunoreactive for TuJ1 (79.49% ± 7.36 and 86.74% ± 4.84, respectively, p = .2036, n = 4) (Fig. 2A, 2C). However, a significant increase in the proportion of TH+ colonies was observed in LRP6 ko cells compared to wt cells (68.78% ± 8.95 and 51.02% ± 10.24, respectively, p = .0102*, n = 4, Fig. 2C). Similar to differentiated Wnt1 mESCs, TH+ cells obtained from either LRP6 wt or ko mESCs showed morphologic characteristics typical of mESC-derived DA neurons (Fig. 2B). Western blot analysis showed increased level of TH protein, but not TuJ1 in LRP6 ko cells after 14 days of differentiation, thereby confirming our previous immunocytochemical observations (Fig. 2D). Finally, RT-PCR again showed that whereas both LRP6 wt and ko cells express typical markers of ventral midbrain DA neurons, elevated levels of these markers were observed in the LRP6 ko cells (Fig. 2E).

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Figure 2. Absence of lipoprotein receptor-related protein 6 (LRP6) promotes mouse embryonic stem cell (mESC) differentiation into TH+/TuJ1+ dopaminergic neurons with ventral midbrain characteristics, using a PA6-based protocol. (A): Representative example of TH (red) and TuJ1 (green) immunostaining of LRP6 wild-type (wt) and knockout (ko) colonies after 14 days of differentiation. (B): High magnification of LRP6 wt and ko mESCs following 14 days of differentiation immunostained for TH (red), with Hoechst nuclear counterstaining (blue). (C): Quantification of TH and TuJ1 positive colonies showing an increase of TH+ colonies in LRP6 ko compared to LRP6 wt after 14 days of differentiation (p < .05, n = 4). (D): Western blot analyses showing increased expression of TH in differentiated cells lacking LRP6, compared to LRP6 wt cells. (E): Reverse transcription-polymerase chain reaction showing expression of dopaminergic markers in differentiated LRP6 wt and ko cells. Sample from PA6 cells was used as negative control. Abbreviations: DAT, dopamine transporter; ko, knockout; LRP6, lipoprotein receptor-related protein 6; TH, tyrosine hydroxylase; wt, wild-type. TuJ1, bIII-tubulin; Foxa2, forkhead box transcription factor A2; Lmx1a, LIM homeobox transcription factor 1a; Nurr1, nuclear receptor related transcription factor 1; Pitx3, Paired-like homeodomain transcriptionfactor 3.

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Increased Neuronal and Dopaminergic Differentiation of Wnt1 Knockout mESCs in Feeder-Free Cultures

We then examined whether the increased number of TH and TuJ1-positive cells in Wnt1/LRP6 mESC cultures was contributed to by a factor or factors derived from the PA6 cells. We therefore used a feeder-free protocol (see Materials and Methods for details) to differentiate Wnt1 ko and wt mESCs for 8 days, and we examined TuJ1 and TH expression by immunochemistry. Interestingly, an increase in the number of TuJ1+ colonies was detected in Wnt1 ko cells compared with wt (Fig. 3A, 3B). Similarly, the lack of Wnt1 significantly increased the number of TH+ colonies (Fig. 3A, 3B), and the number of TH+ cells within the colonies (Fig. 3A). These results indicate that the increases in TuJ1+ and TH+ colonies were not related to the presence or absence of the PA6 feeders, but rather were the direct effect of Wnt1 ablation in mESCs.

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Figure 3. Absence of Wnt1 promotes differentiation of mouse embryonic stem cells (mESCs) into TH+/TuJ1+ dopaminergic neurons with ventral midbrain characteristics independently of stromal cells. (A): Representative example of TH (red) and TuJ1 (green) immunostaining with Hoechst nuclear counterstaining (blue) of Wnt1 wt and ko mESCs after 8 days of feeder-free differentiation in the presence (+P) or the absence (−P) of Shh, FGF8 and bFGF, during days 1-5 of differentiation. (B): Quantification of feeder-free differentiation of Wnt1 mESCs into TuJ1 and TH positive colonies (n = 4), showing an increase in TuJ1 and TH positive colonies in Wnt ko cells and a further increase in TH positive colonies by +P. (C): Reverse transcription-polymerase chain reaction showing expression of dopaminergic markers in differentiated Wnt1 wild-type (wt) and knockout (ko) cells after 12 days of differentiation in the presence (+P) or the absence (−P) of Shh, bFGF, FGF8 treatment. Abbreviations: DAT, dopamine transporter; ko, knockout; LRP6, lipoprotein receptor-related protein 6; TH, tyrosine hydroxylase; wt, wild-type TuJ1, bIII-tubulin; Foxa2, forkhead box transcription factor A2; Lmx1a, LIM homeobox transcription factor 1a; Nurr1, nuclear receptor related transcription factor 1; Pitx3, Paired-like homeodomain transcription factor 3; Shh, sonic hedgehog; FGF8, fibroblast growth factor 8; bFGF, basic fibroblast growth factor.

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We next examined whether the number of TuJ1+ colonies was affected by the treatment of feeder-free mESC cultures with patterning factors (+P, Shh/FGF8/bFGF). No difference was detected following the addition of these factors in either Wnt1 wt (−P: 75.28% ± 6.61 vs. +P: 68.33% ± 5.03) or Wnt1 ko cells (−P: 99.04% ± 0.96 vs. +P: 95.80% ± 2.79). Similarly, treatment of Wnt1 wt cells with patterning factors did not affect the number of TH+ colonies (−P: 5.23% ± 2.25 and +P: 6.33% ± 3.02). However, when Wnt1 ko cells were treated with Shh, FGF8, and bFGF, a significant increase in the number of TH+ colonies was detected (+P: 65.26% ± 6.71, compared to −P: 35.24% ± 5.16) (Fig. 3A, 3B). Similar results were obtained after 14 days of differentiation in feeder-free cultures (data not shown), suggesting that the loss of Wnt1 facilitates the effects of Shh, FGF8, and bFGF on DA differentiation of mESCs. We thus examined Wnt1 wt and ko feeder-free mESC cultures after 12 days of DA differentiation and analyzed the expression of markers typical of ventral midbrain DA neurons by RT-PCR. Expression of TH, Nurr1, Pitx3, Lmx1a, Foxa2, and DAT mRNAs were elevated in Wnt1 ko cells compared with wt cells (Fig. 3C). Interestingly, the expression of DAT was only detected after exposure of mESCs to patterning factors and was increased by deletion of Wnt1, suggesting that the maturation of TH+ cells is enhanced by the patterning factors. Thus, our data suggest that the increase in differentiation of Wnt1 ko mESCs into TuJ1+ and TH+ neurons is not dependent on any factor produced by PA6 cells or added to the cultures, but the number of TH+ cells and their differentiation can be further enhanced by treatment with Shh, FGF8, bFGF.

Neuronal and Dopaminergic Differentiation of mESCs Are Accelerated by Deletion of Lipoprotein Receptor-Related Protein 6 or Wnt1

In order to examine whether the deletion of Wnt1 or LRP6 affected the timing at which DA differentiation takes place in the cultures, we examined Wnt1 ko and LRP6 ko mESCs at earlier time points of differentiation, prior to the appearance of TH+ cells in mESCs cocultured with PA6 cells. Wnt1 wt and ko mESC cultures, seeded at a cell density of 50 cells/cm2, were analyzed at day 7 of differentiation for TH and TuJ1 expression. Typical Wnt1 wt colonies contained very few TuJ1+ cells, and TH+ cells were rarely present (Fig. 4A). On the other hand, typical Wnt1 ko colonies were rich in mature TuJ1+ cells, as shown by the presence of a complex and dense process network, and some of them contained TH+ neurons. In agreement with this, the number of TuJ1+ colonies significantly increased in Wnt1 ko mESC cultures (57.91% ± 7.18) compared with Wnt1 wt cultures (15.58% ± 2.13, p = .0017**, n = 3, Fig. 4B). An increase was also detected in the proportion of TH+ colonies in Wnt1 ko cultures (22.21% ± 6.34) compared to Wnt1 wt (3.58% ± 1.77) after 7 days of differentiation. These differences in the number of TuJ1 and TH positive colonies at early stages of differentiation suggested that deletion of Wnt1 accelerates not only neuronal induction, but also DA differentiation.

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Figure 4. Absence of Wnt1 or LRP6 promotes neurogenesis and DA differentiation in mouse embryonic stem cells (mESCs). (A): Representative picture of Wnt1 knockout (ko) and wild-type (wt) mESCs differentiated on PA6 cells for 7 days and stained for TH (red), TuJ1 (green) and Hoechst (blue). (B): Deletion of Wnt1 increased the percentage of TH and TuJ1 positive colonies, compared to wt (p < .01, n = 3). (C): Day 5: representative example of LRP6 wt and ko mESCs stained for TuJ1 (green) and Hoechst (blue) after 5 days of PA6-based differentiation. See the abundance of rosettes in the LRP6 ko condition. Day 6: immunostaining for TH (red), TuJ1 (green), and nuclear counterstaining with Hoechst (blue) after 6 days of differentiation, showing an increase in the number of TH and TuJ1+ cells. (D): Deletion of LRP6 increased the percentage of TH and TuJ1 positive colonies, compared with wt (p < .05, n = 3). Abbreviations: ko, knockout; LRP6, lipoprotein receptor-related protein 6; TH, tyrosine hydroxylase; TuJ1, βIII-tubulin; wt, wild-type.

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A similar experimental approach was applied to the differentiation of LRP6 mESCs. We first observed that by 5 days of differentiation, more LRP6 ko colonies were rich in rosettes, neuroepithelial structures rarely seen in mESC lines (D.R. and I.L., unpublished observation). Interestingly, TuJ1+ cells were found to delaminate from the rosettes (Fig. 4C). Although LRP6 wt colonies typically had only few TuJ1+ cells and no TH+ cells by day 6 of differentiation, mESCs lacking LRP6 were rich in TuJ1+ cells, possessed a mature morphology (presence of complex processes) and occasionally included a few TH+ cells (Fig. 4C). Interestingly, although only half of the LRP6 wt colonies were TuJ1+ by day 6 of differentiation (52.50% ± 9.02), almost all LRP6 ko colonies had become TuJ1+ (94.76% ± 4.18, p = .0489*, n = 3) (Fig. 4D). This clearly indicates that the absence of LRP6 promotes and accelerates neuronal specification, in a manner similar to Wnt1 ablation. Similarly, TH+ colonies were very rarely found after 6 days in LRP6 wt cultures (1.39% ± 1,389), whereas they were present in LRP6 ko cultures (15.29% ± 4.30, p = .0442*, n = 3). These results indicated that the absence of either LRP6 or Wnt1 promotes both neuronal specification and DA differentiation and accelerates the appearance of DA neurons at an early stage of mESC DA differentiation cultures. Since both Wnt1 and LRP6 are well known components of the Wnt/β-catenin signaling pathway [30, 31], we next investigated whether Wnt/β-catenin signaling was diminished in ko cells.

Decreased Activation of Wnt/β-Catenin Signaling in Lipoprotein Receptor-Related Protein 6 Knockout mESCs

We first examined whether Wnt/β-catenin signaling was decreased in LRP6 ko mESCs compared to wt mESCs in response to increasing doses of Wnt3a, a Wnt ligand shown previously to activate Wnt/β-catenin signaling in dopaminergic cells [32]. Western blot analyses showed a dose-dependent activation of LRP5/6 in wt mESCs in response to Wnt3a, as assessed by phosphorylation of pSer1490 in LRP6 (higher migrating band) and pSer1493 in LRP5 (lower migrating band) (Fig. 5A). Instead, LRP6 ko mESCs showed only phosphorylation of LRP5. Although the absence of LRP6 had no effect on phosphorylation and mobility shift of a Wnt pathway component, Dishevelled-3 (Dvl3), the level of active β-catenin (ABC, a key signaling component of the Wnt/β-catenin pathway) was markedly reduced in both Wnt3a-stimulated and unstimulated LRP6 ko cells compared with their wt counterparts. This is in agreement with a previous report showing LRP6-independent phosphorylation of Dvl [33] and implies a crucial role of LRP6 in Wnt/β-catenin signaling in mESCs.

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Figure 5. Lack of LRP6 abrogates Wnt/β-catenin signaling in mouse embryonic stem cells mESCs. (A): LRP6 wild-type (wt) and knockout (ko) mESCs were stimulated with increasing doses of Wnt3a for 2 hours and analyzed by Western blot. Although Wnt3a stimulation led to similar mobility shift of Dvl3 (compare higher and lower migrating forms of Dvl3 indicated by arrows) in both wt and ko cells, levels of ABC (unphosphorated, activated β-catenin) were diminished in cells lacking LRP6. Stimulation with Wnt3a also induced dose-dependent phosphorylation of LRP6 (arrow) and/or LRP5 (arrowhead). The figure shows a representative example of the three independent experiments. (B): Luciferase assay using SuperTOP/FOP-Flash reporter showing decreased levels of activation in LRP6 ko mESC, in both treated (100 ng/ml Wnt3a) and untreated conditions, compared to wt (p < .05, n = 4, unpaired t test). Abbreviations: Dvl3, dishevelled 3; ko, knockout; LRP6, lipoprotein receptor-related protein 6; wt, wild-type.

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These results were confirmed with luciferase assays using the SuperTOP-FLASH reporter. LRP6 wt mESC cells responded with a 10-fold activation of the SuperTOP-FLASH reporter after a 24-hour treatment with Wnt3a (100 ng/ml). Instead, the control mutated form of the reporter (SuperFOP-FLASH), did not respond to Wnt3a (Fig. 5B). Reporter activation was dramatically reduced in Wnt3a-treated LRP6 ko (0.98 ± 0.32, n = 4), compared with Wnt3a-treated LRP6 wt mESCs (p = .0117*), and was not different from untreated LRP6 ko cells (p = .1201). Interestingly, unstimulated LRP6 ko mESC also showed a significantly lower reporter activation compared with unstimulated LRP6 wt cells (first columns in each condition, Fig. 5B), implying reduced level of endogenous Wnt/β-catenin signaling in LRP6 ko mESCs. All these data provide biochemical evidence that the ability of LRP6 ko mESCs to respond to endogenous or exogenous Wnt ligands and to activate Wnt/β-catenin signaling is impaired.

Decreased Activation of Wnt/β-Catenin Target Genes in Differentiating Wnt1 Knockout mESCs

Since the expression of Wnt1 in undifferentiated wt mESCs is very low (data not shown), Wnt1 ko cells were not expected to exhibit significant biochemical changes in Wnt signaling. However, given that Wnt1 regulates the expression of target genes during development, we reasoned that the lack of Wnt1 could lead to detectable changes in their expression. We first examined the time point at which Wnt1 is upregulated during mESC differentiation and found an increase between days 4 and 5 (Supporting Fig. 2A). We then examined the expression levels of Axin2 and Brachyury, two direct target genes of Wnt/β-catenin pathway [34–36] during days 4 and 5 of Wnt1 mESC differentiation. Interestingly, we found lower levels of Axin2 and Brachyury mRNA in Wnt1 ko mESCs compared with Wnt1 wt mESCs (Supporting Fig. 2B). Thus, our results provide evidence that the absence of Wnt1 impairs Wnt/β-catenin signaling in mESCs during differentiation.

Treatment of mESCs with Dickkopf 1 Promotes Dopaminergic Differentiation

Next, we endeavored to examine whether the effects on neuroectodermal and DA differentiation observed in Wnt1 or LRP6 ko mESCs could be mimicked by treatment of wt mESCs with a known Wnt/β-catenin pathway inhibitor, such as Dickkopf 1 (Dkk1). For this purpose, we used 100 ng/ml of Dkk1, a dose that attenuates Wnt/β-catenin signaling in the SN4741 dopaminergic cell line [32].We then examined R1 mESCs after differentiation for 12 days in a feeder-free protocol and after Dkk1 treatment either from day 1 to day 5 (data not shown) or from day 1 to day 12 (Fig. 6A). Whereas treatment with Dkk1 from day 1-5 of differentiation showed a nonsignificant increase in TH and TuJ1 mRNA expression, treatment with Dkk1 between days 1-12 clearly increased the numbers of TH+ cells (Fig. 6B). The proportion of TH+ colonies significantly increased in Dkk1-treated cultures (54.06% ± 4.37) compared with vehicle-treated control cells (32.18% ± 1.679, p = .0415*, n = 3) (Fig. 6C). Interestingly, the effects of Dkk1 (days 1-12) were detected even in the presence of Shh, FGF8, and bFGF (+P, days 1-5, Fig. 6A), indicating that the effects of Dkk1 are additive with those of these factors. Overall, these data are in agreement with our observations obtained from mESCs lacking Wnt1 or LRP6 and provide evidence that the differentiation of mESCs into TuJ1+ and TH+ neurons can be efficiently enhanced by inhibition of Wnt/β-catenin signaling. Nonetheless, the effects of Dkk1 on differentiation were less robust than the absence of Wnt1 or LRP6 in mESCs. We thus examined the mechanism by which Dkk1 blocked the basal and Wnt3a-induced activation of Wnt/β-catenin pathway. We found that Dkk1 dose dependently decreased both the basal and the Wnt3a-induced activation of Wnt/β-catenin signaling in SN4741 cells and in mESCs (Supporting Fig. 3A and 3B). However, we also found that the effects of Dkk1 (100 ng/ml) were competed in a dose-dependent manner and blocked by 100 ng/ml of Wnt3a (high doses of Dkk1 were needed to overcome the effect of Wnt ligand), showing that the levels of ABC (active β-catenin) depended on the balance between Wnt and Dkk1. Consistent with this idea, all our results indicate that decreases in Wnt/β-catenin signaling by Dkk1 treatment or deletion of Wnt1 or LRP6, increase DA differentiation of mESCs.

thumbnail image

Figure 6. Dickkopf1 (Dkk1) treatment promotes differentiation of mouse embryonic stem cells (mESCs) into TH+/TuJ1+ dopaminergic neurons in a feeder-free protocol. (A): Schematic drawing of differentiation protocol, Dkk1 was present during 11 days of mESC differentiation. Shh, FGF8 and bFGF (+P) were administered between days 1-5. (B): Representative example of control and Dkk1-treated mouse embryonic stem cells stained for TuJ1 (green) and TH (red) after 12 days of differentiation. (C): Quantification of the numbers of TH and TuJ1 positive colonies showing an increase of TH+ colonies in the Dkk1-treated condition after 12 days of differentiation (p <.05, n = 3). Abbreviations: Dkk1, Dickkopf1; TH, tyrosine hydroxylase; TuJ1, βIII-tubulin. Shh, sonic hedgehog; FGF8, fibroblast growth factor 8; bFGF, basic fibroblast growth factor.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

The differentiation of ESCs into a definitive neuronal subtype, such as DA neurons, is of great interest because of their potential use in cell replacement therapy for the treatment of disorders such as Parkinson's disease. Understanding the signaling events during ESC differentiation is thus essential for the establishment of more efficient protocols to generate DA neurons from ESCs. We and others have previously shown that Wnt signaling is important for different stages of DA neuron development [7–13, 37]. However, the role of Wnt signaling in the DA differentiation of ESC has not yet been examined. In this study, we used mESC deficient in Wnt/β-catenin signaling, lacking either a prototypical ligand, Wnt1, or a receptor, LRP6, to investigate the role of Wnt signaling during DA differentiation of mESCs. Using two differentiation protocols, we found that the absence of Wnt1 or LRP6: 1) greatly promoted neuroectodermal differentiation of mESCs, and 2) improved the DA differentiation of mESCs. Moreover, inhibition of Wnt/β-catenin using recombinant Dkk1 protein also enhanced the DA differentiation of mESCs, thus opening the possibility of using Dkk1 in other protocols to promote the DA differentiation of stem cells.

With regard to the mechanism by which deletion of Wnt1 and LRP6 increased neurogenesis, it is well established that LRP6 mediates Wnt/β-catenin signaling [30, 38–40] and that Wnt1 physically interacts with LRP6 [39] to activate this pathway [31, 41, 42]. In agreement with this, we show here that deletion of LRP6 decreased Wnt/β-catenin signaling in mESCs, deletion of Wnt1 decreased the activation of target genes, and deletion of Wnt1 or LRP6 accelerated the formation of TuJ1+ and TH+ cells and colonies. Evidence in the literature suggests both positive and negative roles of the Wnt pathway in neural specification of mESCs. Otero and colleagues [43] reported that treatment of high-density mESC cultures with Wnt3a conditioned media or overexpression of β-catenin promoted neurogenesis. On the contrary, overexpression of SFRP2 was shown to stimulate production of Sox1+ neural progenitors [44], and similarly, treatment of mESC with Dkk1 was described to increase expression of neuronal markers [45]. Thus, our results showing increased neuralization of mESCs could be explained by an early inhibition of Wnt/β-catenin in mESC cultures. However, clear differences in the number of TH+ colonies and TH expression level or number of TH+ cells with characteristics of ventral midbrain DA neurons were found in Wnt1 or LRP6 knockout cells at early and late stages of differentiation, suggesting that the inhibition of Wnt/β-catenin signaling is also beneficial for DA differentiation. Moreover, Dkk1, an inhibitor of the Wnt/β-catenin signaling pathway [46, 47], mimicked the effects of Wnt1 or LRP6 deletion on DA differentiation of mESCs, although the effects of Dkk1 were less pronounced than in the case of ablation of Wnt1/LRP6. We found that two factors contributed to this observation: 1) Dkk1 and Wnt competed in a dose-dependent manner for LRP5/6 receptors and for the inhibition or activation of Wnt/β-catenin signaling (Supporting Fig. 3A, 3B); and 2) the activity of recombinant Dkk1 decreased over time in culture media, as demonstrated by decreased ability to block Wnt-induced pathway activation (Supporting Fig. 3C). However, all our data combined support a model in which lack or inhibition of Wnt/β-catenin signaling has a promoting effect on neuronal specification and DA differentiation of mESCs.

The finding that Wnt1 ablation enhanced neuronal and DA differentiation of mESCs was unexpected in view of the previous analysis of midbrain development in vivo. Indeed, deletion of Wnt1 has been shown to result in severe developmental defects in the midbrain-hindbrain boundary [10, 11], leading to a severe or complete loss of DA neurons in the developing ventral midbrain [10, 11, 13]. Nonetheless, several explanations could account for our results. First, it is important to note that during differentiation, ESCs are exposed both to factors derived from the ESCs themselves and their progeny, as well as extrinsic signals from the feeders. However, feeder-free culture conditions indicated that the feeder cells do not mediate the increase in differentiation observed in Wnt1 deficient mESCs. Alternatively, a putative compensation may be related to the presence of ESC-derived cell-intrinsic or secreted factors during DA differentiation. These factors may or may not be appropriate for ventral midbrain differentiation, and they could be heterochronic (inappropriately early or late midbrain signals) and heterotopic (inappropriate medio-lateral or antero-posterio signals). Provided the relative asynchrony of the ESC cultures, compared with the in vivo situation, this remains as a plausible explanation for the effects seen in Wnt1 ko cultures. However, given that the phenotype we detected is also observed by deletion of a cell-intrinsic factor such as LRP6, our results suggest that the increase in neurons and DA neurons may be caused by cell intrinsic mechanisms, including a possible deregulation of Wnt signaling in mESC cultures. Our biochemical data provide evidence supporting such a model. The absence of LRP6 greatly disrupted responsiveness to both endogenous and exogenous Wnt ligand, as underscored by Figure 5. Moreover, our observation of a dose-dependent phosphorylation of LRP5 in response to Wnt3a, but a minor or negligible activation of Wnt/β-catenin pathway in the absence of LRP6, suggests that LRP6, but not LRP5, has a major impact on Wnt/β-catenin signaling in mESCs. Thus, both our biochemical and differentiation experiments suggest that the inhibition of β-catenin stabilization, along with the subsequent decrease in its capacity to activate transcription, plays a central role in the emergence of the observed mESC phenotypes.

One question that remains to be answered is why inhibition of Wnt/β-catenin increases neuronal and DA differentiation of mESCs. Here it is important to note that Wnts serve multiple functions in vivo and importantly, several of these functions are regulated both by the balance between the activation of canonical and noncanonical Wnt signaling pathways and by the balance between Wnt and other signaling pathways. As for the balance within Wnt signaling pathways, it is known that Wnt1, a prototypical canonical ligand, positively regulates proliferation of DA progenitors [7, 9, 37]. On the other hand, activation of β-catenin-independent signaling by Wnt5a has been found to promote the differentiation and maturation of DA neurons [7, 8, 17, 48]. Interestingly, inhibition or disruption of one branch of the Wnt pathway (in this case Wnt/β-catenin) has been described to be accompanied by increased signaling toward the other branch (β-catenin-independent or noncanonical signaling) [49–51]. In this regard, we and others have recently shown that LRP5/6 inhibits noncanonical Wnt signaling and that loss of LRP6 leads to augmented noncanonical Wnt signaling [51, 52]. Thus, in light of these results, it is tempting to speculate that Wnt/β-catenin loss of function may contribute to the efficient DA differentiation of mESCs by two mechanisms: 1) by promoting efficient neural induction in the early-stage, and 2) by increasing noncanonical signaling, which promotes efficient terminal differentiation of DA neurons. However, it is also possible that a change in the balance between Wnt signaling and other signaling pathways could also contribute to the observed mESC phenotype. Interestingly, it has been recently reported that a balance between Wnt1 and Shh is required for DA neuron development and that Wnt1 is necessary to inhibit the high levels of Shh and allow DA neurogenesis in the developing VM floorplate [12]. In our study, we show that the expression of DAT, a marker for the presence of mature DA neurons, was detected only after exposure of mESCs to Shh/FGF8/bFGF. This is of interest as we found improved DA differentiation of mESCs by administration of Shh/FGF8/bFGF and loss of Wnt1 (or blocking of Wnt/β-catenin signaling). These results suggest a possible imbalance between Shh and Wnt/β-catenin signaling in mESC cultures compared with the ventral midbrain in vivo. Thus our data, in addition to the current literature, underscore the need of individually adjusting the signaling levels of different morphogens in mESC cultures, including Wnt signaling, in order to optimize biologic activities and ESC differentiation protocols.

One of the implications of our study is that inhibition of Wnt/β-catenin signaling in ESCs may be beneficial for the therapeutic application of ESCs. Indeed, our results indicate that lower levels of Wnt/β-catenin signaling are beneficial for DA differentiation of mESCs, but it remains to be examined whether decreased Wnt/β-catenin signaling could be deleterious for other aspects of DA neuron function. In that regard, we think that strategies involving a transient blockade of Wnt/β-catenin signaling, by Dkk1 or pharmacologic inhibitors may be advantageous compared with permanent genetic modifications of stem cells. Thus, future work will focus on evaluating whether mouse and human ESCs with low Wnt/β-catenin signaling, in addition to expressing appropriate markers, also fulfill functionality and safety criteria (that is, long-term functional integration in vivo in the absence of tumor formation). In favor of such a possibility are the findings that Wnt/β-catenin signaling is involved in proliferation [7–11, 37] and several tumors and cancer forms [53]. Moreover, recent studies have linked increased Wnt/β-catenin signaling with accelerated aging or adult DA neuron cell death [54, 55]. In this context, lower Wnt/β-catenin signaling may result in lower proliferation/tumor-forming potential and increased differentiation/survival. If confirmed, transplantation of DA neurons derived from genetically modified ESCs or administration of pharmacologic inhibitors of the Wnt/β-catenin signaling pathway may become useful tools to treat DA neurons in vivo.

In summary, we hereby show that strategies aiming at inhibiting Wnt/β-catenin signaling during ESC differentiation enhance neuralization and DA differentiation. Moreover, these strategies have the potential to improve the efficiency and safety (by decreasing proliferation) of current differentiation protocols, thereby facilitating the future use of ESCs in cell replacement therapy for Parkinson's disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

We thank Dr. R.T. Moon (University of Washington, Seattle, WA) for SuperTOP-FLASH/ SuperFOP-FLASH plasmids, Dr. G. Minchiotti (Institute of Genetics and Biophysics, Naples, Italy) for providing R1 mESC. We would like to thank Johny Söderlund and Lottie Jansson-Sjöstrand for technical assistance and the members of Arenas lab for stimulating discussions. This work was supported by grants from the European Union (Neurostemcell), Swedish Foundation for Strategic Research (INGVAR and CEDB projects), Swedish Research Council (VR2008:2811 and DBRM), Norwegian Research Council and Karolinska Institute to EA, as well as grants from the Ministry of Education, Youth, and Sports of the Czech Republic (MSM0021622430), Academy of Sciences of the Czech Republic (AVOZ50040507, AVOZ50040702), EMBO Installation Grant and Czech Science Foundation (204/09/0498) to VB. LC was supported by a KID grant (6110/06-225) from Karolinska Institute. DR was supported by the Foundation for Science and Technology from the Portuguese Government (SFRH/BD/24585/2005), CLP was supported by a Human Frontiers Science Program long-term fellowship and National Health and Medical Research Council, Australia, CJ Martin fellowship.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information
  • 1
    Parish CL, Arenas E. Stem-cell-based strategies for the treatment of Parkinson's disease. Neurodegener Dis 2007; 4: 339347.
  • 2
    Ciani L, Salinas PC. WNTs in the vertebrate nervous system: From patterning to neuronal connectivity. Nat Rev Neurosci 2005; 6: 351362.
  • 3
    Ille F, Sommer L. Wnt signaling: Multiple functions in neural development. Cell Mol Life Sci 2005; 62: 11001108.
  • 4
    Gaulden J, Reiter JF. Neur-ons and neur-offs: Regulators of neural induction in vertebrate embryos and embryonic stem cells. Hum Mol Genet 2008; 17: R6066.
  • 5
    Castelo-Branco G, Arenas E. Function of Wnts in dopaminergic neuron development. Neurodegener Dis 2006; 3: 511.
  • 6
    Macdonald BT, Semenov MV, He X. SnapShot: Wnt/beta-catenin signaling. Cell 2007; 131: 1204.
  • 7
    Castelo-Branco G, Wagner J, Rodriguez FJ et al. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc Natl Acad Sci U S A 2003; 100: 1274712752.
  • 8
    Parish CL, Castelo-Branco G, Rawal N et al. Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice. J Clin Invest 2008; 118: 149160.
  • 9
    Panhuysen M, Vogt Weisenhorn DM, Blanquet V et al. Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region. Mol Cell Neurosci 2004; 26: 101111.
  • 10
    McMahon AP, Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 1990; 62: 10731085.
  • 11
    Thomas KR, Capecchi MR. Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 1990; 346: 847850.
  • 12
    Joksimovic M, Yun BA, Kittappa R et al. ( 2009). Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis. Nat Neurosci.
  • 13
    Prakash N, Brodski C, Naserke T et al. A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo. Development 2006; 133: 8998.
  • 14
    Kohn AD, Moon RT. Wnt and calcium signaling: beta-catenin-independent pathways. Cell Calcium 2005; 38: 439446.
  • 15
    Seifert JR, Mlodzik M. Frizzled/PCP signalling: A conserved mechanism regulating cell polarity and directed motility. Nat Rev Genet 2007; 8: 126138.
  • 16
    Semenov MV, Habas R, Macdonald BT et al. SnapShot: Noncanonical Wnt Signaling Pathways. Cell 2007; 131: 1378.
  • 17
    Andersson ER, Prakash N, Cajanek L et al. Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo. Plos One 2008; 3: e3517.
  • 18
    Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci 2003; 116: 26272634.
  • 19
    Bryja V, Bonilla S, Cajanek L et al. An efficient method for the derivation of mouse embryonic stem cells. Stem Cells 2006; 24: 844849.
  • 20
    Bryja V, Bonilla S, Arenas E. Derivation of mouse embryonic stem cells. Nat Protoc 2006; 1: 20822087.
  • 21
    Parisi S, D'Andrea D, Lago CT et al. Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells. J Cell Biol 2003; 163: 303314.
  • 22
    Barberi T, Klivenyi P, Calingasan NY et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003; 21: 12001207.
  • 23
    Ying QL, Stavridis M, Griffiths D et al. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003; 21: 183186.
  • 24
    Parish CL, Parisi S, Persico MG et al. Cripto as a target for improving embryonic stem cell-based therapy in Parkinson's disease. Stem Cells 2005; 23: 471476.
  • 25
    Bryja V, Cajanek L, Pachernik J et al. Abnormal development of mouse embryoid bodies lacking p27Kip1 cell cycle regulator. Stem Cells 2005; 23: 965974.
  • 26
    Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000; 28: 3140.
  • 27
    Danielian PS, McMahon AP. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature 1996; 383: 332334.
  • 28
    Andersson E, Tryggvason U, Deng Q et al. Identification of intrinsic determinants of midbrain dopamine neurons. Cell 2006; 124: 393405.
  • 29
    Kittappa R, Chang WW, Awatramani RB et al. The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age. Plos Biol 2007; 5: e325.
  • 30
    He X, Semenov M, Tamai K et al. LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 2004; 131: 16631677.
  • 31
    Young CS, Kitamura M, Hardy S et al. Wnt-1 induces growth, cytosolic beta-catenin, and Tcf/Lef transcriptional activation in Rat-1 fibroblasts. Mol Cell Biol 1998; 18: 24742485.
  • 32
    Bryja V, Schulte G, Arenas E. Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin. Cell Signal 2007; 19: 610616.
  • 33
    Gonzalez-Sancho JM, Brennan KR, Castelo-Soccio LA et al. Wnt proteins induce dishevelled phosphorylation via an LRP5/6-independent mechanism, irrespective of their ability to stabilize beta-catenin. Mol Cell Biol 2004; 24: 47574768.
  • 34
    Jho EH, Zhang T, Domon C et al. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol 2002; 22: 11721183.
  • 35
    Yamaguchi TP, Takada S, Yoshikawa Y et al. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 1999; 13: 31853190.
  • 36
    Arnold SJ, Stappert J, Bauer A et al. Brachyury is a target gene of the Wnt/beta-catenin signaling pathway. Mech Dev 2000; 91: 249258.
  • 37
    Castelo-Branco G, Rawal N, Arenas E. GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J Cell Sci 2004; 117: 57315737.
  • 38
    Pinson KI, Brennan J, Monkley S et al. An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 2000; 407: 535538.
  • 39
    Tamai K, Semenov M, Kato Y et al. LDL-receptor-related proteins in Wnt signal transduction. Nature 2000; 407: 530535.
  • 40
    Wehrli M, Dougan ST, Caldwell K et al. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 2000; 407: 527530.
  • 41
    McMahon AP, Moon RT. Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis. Cell 1989; 58: 10751084.
  • 42
    Sokol S, Christian JL, Moon RT et al. Injected Wnt RNA induces a complete body axis in Xenopus embryos. Cell 1991; 67: 741752.
  • 43
    Otero JJ, Fu W, Kan L et al. Beta-catenin signaling is required for neural differentiation of embryonic stem cells. Development 2004; 131: 35453557.
  • 44
    Aubert J, Dunstan H, Chambers I et al. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 2002; 20: 12401245.
  • 45
    Verani R, Cappuccio I, Spinsanti P et al. Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid. J Neurochem 2007; 100: 242250.
  • 46
    Semënov MV, Tamai K, Brott BK et al. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol 2001; 11: 951961.
  • 47
    Bafico A, Liu G, Yaniv A et al. Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat Cell Biol 2001; 3: 683686.
  • 48
    Hayashi H, Morizane A, Koyanagi M et al. Meningeal cells induce dopaminergic neurons from embryonic stem cells. Eur J Neurosci 2008; 27: 261268.
  • 49
    Veeman MT, Axelrod JD, Moon RT. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 2003; 5: 367377.
  • 50
    Topol L, Jiang X, Choi H et al. Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation. J Cell Biol 2003; 162: 899908.
  • 51
    Tahinci E, Thorne CA, Franklin JL et al. Lrp6 is required for convergent extension during Xenopus gastrulation. Development 2007; 134: 40954106.
  • 52
    Bryja V, Andersson ER, Schambony A et al. The extracellular domain of Lrp5/6 inhibits noncanonical Wnt signaling in vivo. Mol Biol Cell 2009; 20: 924936.
  • 53
    Clevers H. Wnt/beta-Catenin Signaling in Development and Disease. Cell 2006; 127: 469480.
  • 54
    White BD, Nguyen NK, Moon RT. Wnt signaling: It gets more humorous with age. Curr Biol 2007; 17: R923925.
  • 55
    Rawal N, Corti O, Sacchetti P et al. ( 2009). Parkin protects dopaminergic neurons from excessive Wnt/beta-catenin signaling. Biochem Biophys Res Commun doi:10.1016/j.bbrc. 2009. 07.014.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
STEM_210_sm_suppinfofigure1.tif12814KSupplementary figure 1: (A) Genotyping of wt and ko alleles by PCR showed that all ko mESC lines are homozygotes for the gene of interest. (B) Wnt 1 and LRP6 ko mESC lines showed no apparent differences in colony morphology compared to wt. (C) Western blot analyses showed no difference in expression level of Oct3/4 in Wnt 1 and LRP6 ko mESC lines compared to wt.
STEM_210_sm_suppinfofigure2.tif341KSupplementary figure 2: mRNA expression of Wnt1 (A) and its target genes Axin2 and Brachyury (B) during feeder free differentiation of mESCs. mRNA levels were normalized to their corresponding control conditions: Wnt1 expression in ventral midbrain embryonic day 11.5 (VM E11:5) in A, or in wt mESCs at day 4 of differentiation. Graphs represent summary of at least 4 experiments, * p≤0.05.
STEM_210_sm_suppinfofigure3.tif706KSupplementary figure 3: Characterization of the effects of Dkk1 on Wnt/β-catenin signaling in a dopaminergic cells (A) and mESCs (B and C). (A) Dkk1 decreased the activation of the Wnt/β-catenin pathway. SN4741 cells were treated with increasing doses (ng/ml) of Wnt3a and/or Dkk1 for 2.5 hours and analyzed for level of ABC (unphosphorated, activated β-catenin) and mobility shift of Dvl3 by western blotting. (B) Dkk1 decreased the activation of the Wnt/β-catenin pathway in mESCs treated with Wnt3a. LRP6 wt and ko mESCs were treated for 2.5 hour with Wnt3a (50 ng/ml), and Dkk1 (100ng/ml or 400 ng/ml) or vehicle, as indicated. (C) wt mESCs were analyzed for LRP5/6 phosphorylation and ABC level by western blot. Cells were treated with either 0.1% BSA in PBS for 16 hours (vehicle), 500 ng/ml Dkk1for 16 hours and 75ng/ml Wnt3a during the last 4 hours (Dkk1 pretreatment), or vehicle for 16 hours plus 75ng/ml of Wnt3a and 500 ng/ml of Dkk1 during the last 4 hours (Dkk1 co-treatment). Note the lower ABC and phosho-LRP5/6 levels in the Dkk1 co-treatment compared to Dkk1 pretreatment.
STEM_210_sm_suppinfo.doc23KSupporting Information

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