TY - JOUR
AU - Minchiotti, Gabriella
AB - Abstract Cripto is a glycosylphosphatidylinositol-anchored coreceptor that binds Nodal and the activin type I (ALK)-4 receptor, and is involved in cardiac differentiation of mouse embryonic stem cells (mESCs). Interestingly, genetic ablation of cripto results in increased neuralization and midbrain dopaminergic (DA) differentiation of mESCs, as well as improved DA cell replacement therapy (CRT) in a model of Parkinson's disease (PD). In this study, we developed a Cripto specific blocking tool that would mimic the deletion of cripto, but could be easily applied to embryonic stem cell (ESC) lines without the need of genetic manipulation. We thus screened a combinatorial peptide library and identified a tetrameric tripeptide, Cripto blocking peptide (BP), which prevents Cripto/ALK-4 receptor interaction and interferes with Cripto signaling. Cripto BP treatment favored neuroectoderm formation and promoted midbrain DA neuron differentiation of mESCs in vitro and in vivo. Remarkably, Cripto BP-treated ESCs, when transplanted into the striatum of PD rats, enhanced functional recovery and reduced tumor formation, mimicking the effect of genetic ablation of cripto. We therefore suggest that specific blockers such as Cripto BP may be used to improve the differentiation of ESC-derived DA neurons in vitro and their engraftment in vivo, bringing us closer towards an application of ESCs in CRT. Embryonic stem cells, Neural differentiation, Cell transplantation, Cellular therapy, Parkinson's disease Introduction Cripto is a glycosylphosphatidylinositol (GPI)-anchored coreceptor that belongs to the Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) protein family and plays important roles during embryonic development, stem cell function, and cancer progression [1, 2]. Cripto binds to ligands of transforming growth factor beta (TGF-β) family such as Nodal and Nodal-related growth differentiation factor-1 or -3, and interacts with the activin type I (ALK-4) receptor, thereby activating a complex formed with Activin type IIB serine/threonine kinase receptor [3–5]. Upon receptor activation, the intracellular effectors Smad2/Smad3 become phosphorylated and together with Smad4 translocate into the nucleus to regulate gene expression [6]. Cripto is thus a modulator of TGF-β signaling that inhibits activin signaling [7, 8] and the antiproliferative effects of TGFβ [9–11]. Consistent with that, we previously showed that Cripto acts through the Nodal/ALK-4 pathway to negatively regulate neural differentiation and permit the entry of embryonic stem cells (ESCs) into cardiac lineage [12]. In support of these findings, disruption of cripto in mouse embryonic stem cells (mESCs) enhanced neurogenesis and midbrain dopaminergic (DA) differentiation in vitro [13, 14]. These results suggested that Cripto blocking could be used as a tool to improve the efficiency of neural differentiation protocols for cell replacement therapy (CRT) in neurodegenerative disorders such as Parkinson's disease (PD) [15]. PD has been identified as a priority target for CRT because the classical signs of the disease (tremor, rigidity, and hypokinesia) are caused by the progressive degeneration of a circumscribed cell population, the substantia nigra DA neurons. The idea of using stem cells for CRT in PD patients has been fueled by the finding that human ventral midbrain fetal tissue grafts, under specific conditions, improve the symptoms and reduce the need of levodopa in these patients [16, 17]. Pluripotent stem cells have become an attractive cell source for CRT because they can be easily expanded and they can give raise any cell type in an organism. Thus, much work has focused on the development of methods for generating midbrain DA neurons from ESCs [18, 19]. However, a major remaining challenge in the field is the need to improve the functional outcome and the safety of ESC grafting [20, 21]. This has lead to the development of cell sorting strategies to eliminate unwanted cells and/or select the desired cell types [22, 23], thus reducing or eliminating uncontrolled growth of ESCs and tumor formation [24–27]. Other strategies have focused on reducing the presence of pluripotent cells and proliferating neural progenitors by gaining further control over critical developmental processes such as neural induction and neuronal differentiation. In particular, inhibition of bone morphogenetic protein (BMP) signaling with Noggin [28, 29], or a broader inhibition of Lefty, activin and TGFβ signaling, with Noggin and SB431542, have been reported to reduce the presence of pluripotent cells and increase neural differentiation in ESC preparations [28–30]. Interestingly, transplantation of Noggin-treated human Embryonic Stem (hES)-derived DA cells lead to engraftment of TH+ cells and behavioral improvements in hemiparkinsonian rats, but they also lead to tumor/outgrowth formation [28, 29]. Previous results from us and others have shown that deletion of cripto in mESCs, increases DA differentiation [13, 14]. Although transplantation of cripto−/− mESCs-derived embryoid body (EBs) cells at high density lead to tumor formation [14], when grafted at low density, they abolished tumor/outgrowth formation by ESCs in vivo, and promoted functional recovery in parkinsonian rats [13]. This finding suggested that strategies aiming at achieving efficient Cripto blocking, other than genetic modification, could be broadly used to improve the DA differentiation of different pluripotent cells. We therefore endeavored to develop a compound capable of inhibiting Cripto signaling in ESCs, with the goal of using such a tool to enhance DA differentiation and improve functional recovery after transplantation in animal models of PD. For this purpose, we screened a random combinatorial tetrameric tripeptide library built with non-natural amino acids on a multimeric scaffold, which ensured enhanced recognition capabilities deriving from the large recognition surface and the high flexibility of the final compounds [31]. Peptide mixtures composing the library were utilized as competitors of Cripto/ALK-4 interaction in an enzyme-linked immunosorbent assay (ELISA)-based competition assay, following an iterative process. Selected peptides were evaluated for their ability to inhibit Cripto-induced Smad2 phosphorylation in ESCs and promote robust neuronal differentiation. These assays allowed us to identify a tetrameric tripeptide, named Cripto blocking peptide (Cripto BP). The action/activity of this peptide was further tested on DA differentiation of mESCs in vitro. Moreover, Cripto BP-treated mESCs were transplanted in parkinsonian rats to examine their ability to promote functional recovery and reduce tissue overgrowth or tumor formation. Materials and Methods Iterative Deconvolution of the Tetrameric Tripeptide Library The screening was performed by an ELISA assay (supporting information text). In the first step of the deconvolution process, the peptide pools were tested with a molar excess of 30,000-fold (calculated for each single peptide) over Cripto (8.6 × 10−10 M; Fig. S1). Once identified an active pool(s), a dose-dependent inhibition using 200-, 500-, 1,000-, 5,000-, 12,500- and 30,000-fold excess was performed to confirm the inhibitory property (Fig. S2). The first active pool selected was that labeled as 4-23-X (X indicated random positions), where “23” identified the amino acid L-cysteine(S-benzyl) [L-Cys(Bzl)] (Fig. S2). This peptide pool was resynthesized as 30 single peptides and submitted to the second screening using a molar excess of 1,000-fold (calculated for each single peptide) over Cripto; the screening allowed the identification of the peptide 4-23-23 as the best performing in our test (Fig. S3). To finally investigate the N-terminal position, where D-Glu was initially arbitrarily placed, 30 single peptides labeled as B-23-23 were synthesized and submitted to the final screening (Figs. S4, S5). The peptide indicated as 26-23-23, where “26” identified the amino acid L-methionine-sulfoxide [L-Met(O)], was the molecule showing the optimal inhibitory activity. The activity of this peptide was further confirmed using the high-performance liquid chromatography (HPLC) purified molecule. ESC Culture and Differentiation Mouse embryonic stem (ES) cell lines, both wild-type (R1 [32]) and cripto−/− [33], were used throughout the study and were cultured as previously described [34]. For in vitro cardiac differentiation as shown in Figure 3, ESCs were aggregated as EBs in 15% fetal bovine serum (FBS) as previously described [12, 34]. For the dopaminergic differentiation (Fig. 4A), ESCs were seeded at low density onto a stromal feeder layer for 14 days in the presence of sequential morphogens and mitogens (sonic hedgehog [Shh], fibroblast growth factor 2 [FGF2], FGF-8) as well as peptides (control peptide [CP] or Cripto BP). See supporting information text for details. Western Blotting and Smad2 Phosphorylation Assay Two-day-old cripto−/− mESC-derived EBs were serum starved for 3 hours in Dulbecco's modified Eagle's medium (GIBCO, Invitrogen, Carlsbad, CA, http://www.invitrogen.com/site/us/en/home/brands/Gibco.html?CID=fl-gibco) without leukemia inhibitory factor (LIF) and in low serum (0.5% FBS). Following starvation, EBs were incubated for 20 minutes at 37°C with recombinant mouse Cripto protein ([12]; 0.5 μg/ml) either alone or in the presence of the indicated amount of peptides. Anti-Smad2 and anti-phospho-Smad2 (Ser465/467) Antibodies (Cell Signaling Technology, Denvers, MA, http://www.cellsignal.com/) were used, following the manufacturer's instructions. RNA Preparation and Quantitative Reverse Transcription Polymerase Chain Reaction Total RNAs was extracted with TRIzol kit (Life Technologies Inc., Carlsbad, CA, http://www.lifetechnologies.com/about-life-technologies/contact-us.html) according to manufacturer's instructions and quantitative polymerase chain reaction (QPCR) was performed using SYBR Green PCR master mix (EuroClone, Milano, IT, http://www.euroclonegroup.it/), according to manufacturer's instructions. Primers are described in supporting information Table 2. Determination of In Vitro Dopamine and Dihydroxphenylacetic Acid Levels Dopamine release and turnover was determined in vitro using reverse phase HPLC. DA turnover was expressed as the concentration of dihydroxphenylacetic acid (DOPAC) to DA [DOPAC/DA]. Analysis was performed on triplicate wells on three independent differentiation experiments. 6OHDA Lesioning and Transplantation of ESCs All experiments were performed according to the guidelines of the European Community and were approved by the local ethical committee. Sprague-Dawley rats (250–350 g; from ARC, Australia) were housed and treated according to the guidelines of the Australian and Howard Florey Institute animal ethics committee (07-030). Thirty-seven animals received unilateral stereotaxic injections of 6-Hydroxydopamine (OHD) into the substantia nigra pars compacta (SNpc) to create a complete lesion of the midbrain DA neurons, as previously described [13]. Lesioned animals were selected for transplantation based on their response to amphetamine-induced rotational behavior as previously described [13]. Animals making greater than six rotations/minute (i.e., lesions greater than 80%) were selected for grafting. Two days prior to grafting animals began cyclosporine-A immune suppression (10 mg/kg). Fourteen days postlesioning selected animals (based on behavioral rotations) were again anesthetized and stereotaxically injected with 1 μl of cell suspension at each of the four sites. Stereotaxic coordinates of first and second injection (from Bregma): anterior 0.2 mm, lateral 3.0 mm, ventral 4.5 and 5.0 mm; third and fourth injection, anterior 1.0 mm, lateral 2.5 mm, ventral 4.5 and 5.0 mm; incisor bar 0 mm [35]). Wild-type mESCs were differentiated as outlined in Fig. S6a, including either CP or Cripto BP; media was also supplemented with Noggin (300 ng/ml, day 0–8, to enhance neuronal induction) as well as Wnt5a (200 ng/ml, day 8–11), to promote DA neuron differentiation. Following in vitro differentiation, ESC colonies were dissected away from the stromal feeder layer and dissociated using collagenase/dispase with agitation (700 μg/ml, Roche, for 20 minutes, 80 rpm). Cells were subsequently resuspended at a density of 3,000 cells/μl in preparation for grafting. Five rats received Sham injections (4 μl N2 media, 1 μl/site), 16 rats received a total of 12,000 mESCs-derived cells (4 × 1 μl grafts) pretreated with the CP and a further 16 rats received grafts pretreated with the Cripto BP. Animals were tested for functional improvements at 2, 4, 6, and 8 weeks post-transplantation by amphetamine-induced rotational behavior. Eight weeks postgrafting, rats were killed by an overdose of sodium pentobarbital (100 mg/kg i.p.) and intracardially perfused as previously described [13]. Brains were removed, postfixed for 1 hour, and then left overnight at 4°C in 20% sucrose in phosphate-buffered saline (PBS). The following day, 20-μm-thick coronal sections were cut serially through the striatum (12 series) and 50 μm through the SNpc and mounted directly onto chrom alum gelatinized slides. Histological Analysis Immunofluorescence analysis were performed as previously described [12, 13]. The following primary antibodies were used: mouse anti-βIII-tubulin (Tuj1, 1:1,000, Promega, Madison, WI, http://www.promega.com); rabbit anti-tyrosine hydroxylase (TH; 1:250, PelFreez, Rogers, AR, http://www.pelfreez-bio.com/); sheep anti-TH (1:250, PelFreez, Rogers, AR, http://www.pelfreez-bio.com/); Nurr-1 (1:500, Santa Cruz, CA, USA, http://www.scbt.com/); rabbit anti-Pitx3 (1:200, a gift from P. Burbach, Rudolf Magnus Institute of Neuroscience, Utrecht), rabbit anti-GIRK-2 (1:100, Chemicon, Temecula, CA, http://www.millipore.com/company/cp1/redirect-ab), rabbit anti-GABA (1:500, Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com/customer-service.html); rabbit anti-5HT (1:200, Biogenesis Ltd, Dorset, UK, www.biogenesis.co.uk/); rabbit anti-NET (1:20, Chemicon; rabbit anti-cytokeratin (1:4,000, DAKO, Glostrup, DK, http://www.dako.com/); rat anti-CD29 (1:200, BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/), rabbit anti-Ki67 (Neomarkers Thermofisher, CA, USA, http://www.thermofisher.com/global/en/about/home.asp), mouse anti-nestin (1:200, Chemicon, Temecula, CA, http://www.millipore.com/company/cp1/redirect-ab); mouse anti-Oct4 (1:100, Santa Cruz, Santa Cruz, CA, USA, http://www.scbt.com/), rabbit anti-glial fibrillary acidic protein, (GFAP, 1:200, DAKO, Glostrup, DK, http://www.dako.com/). Appropriate fluorophore conjugated secondary antibodies: (Cyanin-2, Cy3, and Cy5, 1:200, Jackson ImmunoResearch, Suffolk, UK, http://www.jireurope.com/home/contact.asp) were used for visualization. Labeling was visualized by light or fluorescent illumination using an inverted microscope (DMIRB, Leica Microsystems, Wetzelar, DE, http://www.leica-microsystems.com) or upright microscope (Leica DMLB2). Images were acquired on a DC 350 FX camera (Leica). The grading analysis (Fig. 3) was performed blinded by two investigators; to get a reliable unbiased analysis, all the EBs formed in suspension culture were individually plated in 48 multiwell plates and used for morphological (Fig. 3A, 3B) and immunofluorescence analysis (Fig. 3C, 3D). The proportion of neuron containing colonies (labeled using the immature neuronal marker βIII-tubulin, Tuj1) and TH neuron containing colonies were counted and expressed as a percentage of total colonies (Fig. 4). The total number of dopaminergic neurons/μm2 colony (identified using a marker for the precursor enzyme in DA synthesis, TH) were also counted (Fig. 4). Experiments were performed in triplicate. Semiquantitative scoring was employed to estimate the cell composition of the tumors, as previously described [14]. Tumors were stained and scored by blinded investigators using the following scales: 0 = no cells (0%); 1 = few cells (<5%); 2 = moderate cells (5–20%); 4 = dominant cell type (>50%). One series of slides containing grafted striata was counterstained using 1% Neutral red to visualize the graft/tumor size and the gross morphology of the striatum. Area of grafts and tumors was calculated using Stereo Investigator software (MicroBrightField, VT). Statistical Analysis Values are expressed as means SEM or SD. The statistical significance of difference was determined by ANOVA followed by Tukey's post hoc test or a Student's t-test. Ranking of in vitro differentiation (Fig. 3C) was performed using a Wilcoxon rank sum test. Statistical Package for Social Sciences software package, version 11.0 SPSS, Chicago, IL and Sigma Stat 3.0 software were used for statistical analysis with significance set at p < .05. Results Identification of a Novel Peptide that Antagonizes Cripto/ALK-4 Receptor Interaction To identify Cripto/ALK-4 antagonists capable of disrupting downstream signaling, we screened a tetrameric tripeptide library synthesized using 29 non-natural amino acids to increase peptide stability, plus Glycine, (supporting information Table 1). To increase the molecular surface, the library was assembled as previously described [31] on a peptide scaffold composed of a poly-lysine core [36]. To facilitate the characterization of the peptide mixtures, and at the same time to expedite the screening procedure, the first residue was arbitrarily fixed. A negatively charged amino acid, the D-glutamic acid (D-Glu, denoted 4; Fig. 1A and supporting information Table 1), was thus initially chosen as the common, N-terminal residue, to favor peptide solubilization. The library was arranged in 30 pools, each of them composed of 30 tetrameric tripeptides, which differ for the amino acid (denoted B) on the second position (Fig. 1A), while the residue in the third position (denoted X) was randomly added. The total complexity of the library was therefore of 302 = 900 different peptides. The screening was carried out by an ELISA-based competition assay whereby the displacement of soluble recombinant Cripto from coated ALK-4 by the peptide pools was evaluated. The iterative procedure consisted of three screening rounds, which led to the selection of the 26-23-23 tetramer formed by L-methionine-sulfoxide [L-Met(O)] (denoted as 26) in position 1, and S-benzylated L-cysteine [Cys(Bzl)] (denoted as 23) in positions 2 and 3 of the tripeptide (Fig. 1A), (for details see supporting information Text and Figs. 1–4). The selected tetrameric tripeptide (26-23-23, supporting information Fig. 5) was thereafter named Cripto BP. This peptide blocked the interaction of Cripto and ALK-4 with an estimated IC50 of 400 nM, while the control tetrameric Peptide (CP), D-Ser-Ala-Cha, and a dimeric peptide (DP) variant of the 26-23-23 peptide had no effect (Fig. 1B). We thus performed ELISA assays to evaluate whether Cripto BP- or CP-coated to microtiter plates could bind recombinant Cripto and/or ALK-4 receptor. Cripto but not ALK-4 specifically recognized the immobilized Cripto BP, in a dose-dependent manner (Fig. 1C). Neither Cripto nor ALK-4 recognized the CP, confirming the specificity of Cripto BP for Cripto binding. 1 Open in new tabDownload slide (A): Schematic representation of the iterative screening strategy. (1) NH2-D-Glu (D-glutamic acid, also denoted as “4”, see supporting information Table 1) is the common N-terminal residue, (B) identifies the fixed residue, which denotes 1 of 30 amino acids (aa1-30, supporting information Table 1) and X indicates random positions (4-B(1–30)-X). The total complexity of the first library was therefore of 302 = 900 different peptides. In the first screening round (1), 4.23.X identifies the first active pool, where “23” indicates L-cysteine(S-benzyl) [L-Cys(Bzl)]. In the second round of screening (2) the pool 4-23-X was re-synthesized in 30 single peptides (4-23-B(1–30)), and the residue “23” was identified in the third position (4-23-23). Finally, 30 single peptides (B(1–30)-23-23) were submitted to the third screening round (3), which identified the residue “26,” where “26” indicates L-methionine-sulfoxide [L-Met(O)]. The 26-23-23 peptide is also referred to as Cripto BP. (B) Dose-dependent inhibition of Cripto/ALK-4 interaction exerted by Cripto BP, CP (i.e., tetrameric tripeptide D-Ser-Ala-Cha) and dimeric peptide (DP; i.e., dimeric tripeptide 26-23-23) in ELISA-based competition assays (n = 3). (C): Dose-dependent interaction of Cripto or ALK-4 to Cripto BP. The indicated peptides (BP and CP) were coated (50 μM) on the plates and increasing amounts of either Cripto or ALK-4 receptor were used in binding, ranging from 50 to 200 ng/ml. Cripto but not ALK-4 bound Cripto BP; whereas, both Cripto and ALK-4 failed to bind the CP. Mean ± SD, *, p < .0005. Abbreviations: ALK-4, activin type I receptor; BP, blocking peptide; CP, control peptide; CRIPTO, Cripto; ELISA, enzyme-linked immunosorbent assay. 1 Open in new tabDownload slide (A): Schematic representation of the iterative screening strategy. (1) NH2-D-Glu (D-glutamic acid, also denoted as “4”, see supporting information Table 1) is the common N-terminal residue, (B) identifies the fixed residue, which denotes 1 of 30 amino acids (aa1-30, supporting information Table 1) and X indicates random positions (4-B(1–30)-X). The total complexity of the first library was therefore of 302 = 900 different peptides. In the first screening round (1), 4.23.X identifies the first active pool, where “23” indicates L-cysteine(S-benzyl) [L-Cys(Bzl)]. In the second round of screening (2) the pool 4-23-X was re-synthesized in 30 single peptides (4-23-B(1–30)), and the residue “23” was identified in the third position (4-23-23). Finally, 30 single peptides (B(1–30)-23-23) were submitted to the third screening round (3), which identified the residue “26,” where “26” indicates L-methionine-sulfoxide [L-Met(O)]. The 26-23-23 peptide is also referred to as Cripto BP. (B) Dose-dependent inhibition of Cripto/ALK-4 interaction exerted by Cripto BP, CP (i.e., tetrameric tripeptide D-Ser-Ala-Cha) and dimeric peptide (DP; i.e., dimeric tripeptide 26-23-23) in ELISA-based competition assays (n = 3). (C): Dose-dependent interaction of Cripto or ALK-4 to Cripto BP. The indicated peptides (BP and CP) were coated (50 μM) on the plates and increasing amounts of either Cripto or ALK-4 receptor were used in binding, ranging from 50 to 200 ng/ml. Cripto but not ALK-4 bound Cripto BP; whereas, both Cripto and ALK-4 failed to bind the CP. Mean ± SD, *, p < .0005. Abbreviations: ALK-4, activin type I receptor; BP, blocking peptide; CP, control peptide; CRIPTO, Cripto; ELISA, enzyme-linked immunosorbent assay. Cripto BP Antagonizes Cripto Signaling, Impairing Cardiomyogenesis, and Promoting Neurogenesis in mESCs Given the neutralizing activity of the peptide, we first asked whether Cripto BP was able to block Smad2-dependent Cripto signaling in mESCs. We thus evaluated whether Cripto BP was able to prevent Cripto-induced Smad2 phosphorylation of serum starved 2-day-old cripto−/− EBs. Recombinant soluble Cripto protein stimulated Smad2 phosphorylation, as assessed by Western blot analysis ([12] and Fig. 2); this effect was not modified by the presence of increasing amounts of either Cripto DP or CP (2.5 μM and 25 μM; Fig. 2). On the contrary, Cripto-induced Smad2 phosphorylation was inhibited by Cripto BP and completely blocked at 25 μM (125× molar excess; Fig. 2). We then assessed whether Cripto BP was able to antagonize the endogenous Cripto signaling and thus mimicking the effect of genetic ablation of cripto in a mESC differentiation assay, that is, blocking cardiomyogenesis and inducing neuronal differentiation, under the well-established conditions, which promote cardiac differentiation of mESCs [12, 37]. Two day-old wild-type EBs were treated with increasing amounts of either CP or Cripto BP, ranging from 5.0–25 μM, and the percentage of EBs containing beating areas was scored from day 8 to 12 of the differentiation (n = ∼60 EBs/group; Fig. 3B). Given that the peptides were dissolved in dimethyl sulfoxide (DMSO), control untreated EBs were compared with DMSO-treated EBs. Interestingly, neither DMSO, the tetrameric CP (Fig. 3B), nor the DP (data not shown) affected the percentage of beating EBs. Instead, treatment of wild-type EBs with Cripto BP, induced a dose-dependent inhibition of cardiomyogenesis, as shown by the progressive reduction of rhythmically contracting EBs (at 25 μM, BP 3.7% ± 3.5 vs. CP 82.9% ± 11.17, **, p < .0001; Fig. 3B). 2 Open in new tabDownload slide Cripto BP prevents Cripto-induced Smad2 phosphorylation. Two-day-old cripto−/− mouse embryonic stem cells-derived embryoid bodies were stimulated with recombinant Cripto protein (0.5 μg/ml) either alone or in the presence of increasing amount (2.5 μM and 25 μM) of the peptides: DP (dimeric 26-23-23), CP (control tetrameric D-Ser-Ala-Cha) and BP (tertrameric 26-23-23), or left untreated as control. Cripto BP, but not CP or DP inhibits Cripto-induced Smad2 phosphorylation, as shown by Western blot, using anti-phospho-Smad2 antibodies. Levels of total Smad2 were also compared. Smad2 phosphorylation was expressed as ratio between arbitrary densitometric units of P-Smad2 and Smad2. Data are mean ± SEM, n ≥ 3. Abbreviations: BP, blocking peptide; CP, control peptide; DP, dimeric peptide. 2 Open in new tabDownload slide Cripto BP prevents Cripto-induced Smad2 phosphorylation. Two-day-old cripto−/− mouse embryonic stem cells-derived embryoid bodies were stimulated with recombinant Cripto protein (0.5 μg/ml) either alone or in the presence of increasing amount (2.5 μM and 25 μM) of the peptides: DP (dimeric 26-23-23), CP (control tetrameric D-Ser-Ala-Cha) and BP (tertrameric 26-23-23), or left untreated as control. Cripto BP, but not CP or DP inhibits Cripto-induced Smad2 phosphorylation, as shown by Western blot, using anti-phospho-Smad2 antibodies. Levels of total Smad2 were also compared. Smad2 phosphorylation was expressed as ratio between arbitrary densitometric units of P-Smad2 and Smad2. Data are mean ± SEM, n ≥ 3. Abbreviations: BP, blocking peptide; CP, control peptide; DP, dimeric peptide. 3 Open in new tabDownload slide Cripto BP prevents cardiomyogenesis and induces neurogenesis in wild-type ESCs. (A): In vitro cardiac differentiation scheme of wild-type ESCs. (B): Cripto BP (but not CP or DMSO) prevented cardiomyogenesis. Peptides were added to 2-day-old wild-type mouse ESCs-derived EBs; the percentage of EBs with rhythmically contracting areas was scored on day 13 (n = ∼60 EBs/group; data are mean ± SD; *, p < .001, **, p < .0001). (C): Grades of neuronal differentiation of wild-type EBs, as arbitrarily defined. Grade 0: absence of neurons; Grade 1: few, isolated neurons immunoreactive to β-III tubulin antibodies; Grade 2: small areas of β-III tubulin-ir cells; Grade 3: dense network of β-III tubulin-ir cells. Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. Scale bar (A–C) = 100 μM, (D) = 75 μM in. (D): BP (but not CP or DP) promoted robust neuronal differentiation in wild-type EBs. Two-day-old wild-type EBs were treated with 25 μM of the indicated peptides and immunofluorescence analysis was performed on day 13 (n = ∼60 EBs/group, data are mean ± SD; **, *, p ≤ .03). The degree of neuronal differentiation was scored according to panel (C). (E): Gene expression analysis of mesoderm and neuronal markers. Two-day-old wild-type EBs were treated either with the indicated peptides at 25 μM (CP; BP) or vehicle (DMSO) and the expression profile of mesoderm/cardiac and neuronal markers was analyzed on day 13, by quantitative reverse transcription polymerase chain reaction. mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and presented as fold change in gene expression relative to the control (DMSO = 1). Data are mean ± SD, n ≥ 3; *, p < .05 **, p < .01. Abbreviations: AFP, alpha-fetoprotein; BP, blocking peptide; CP, control peptide; DAT, dopamine transporter; DMSO, dimethyl sulfoxide; DP, dimeric peptide; EB, embryoid body; ESC, embryonic stem cell; NFM, neurofilament medium polypeptide; TH, tyrosine hydroxylase. 3 Open in new tabDownload slide Cripto BP prevents cardiomyogenesis and induces neurogenesis in wild-type ESCs. (A): In vitro cardiac differentiation scheme of wild-type ESCs. (B): Cripto BP (but not CP or DMSO) prevented cardiomyogenesis. Peptides were added to 2-day-old wild-type mouse ESCs-derived EBs; the percentage of EBs with rhythmically contracting areas was scored on day 13 (n = ∼60 EBs/group; data are mean ± SD; *, p < .001, **, p < .0001). (C): Grades of neuronal differentiation of wild-type EBs, as arbitrarily defined. Grade 0: absence of neurons; Grade 1: few, isolated neurons immunoreactive to β-III tubulin antibodies; Grade 2: small areas of β-III tubulin-ir cells; Grade 3: dense network of β-III tubulin-ir cells. Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. Scale bar (A–C) = 100 μM, (D) = 75 μM in. (D): BP (but not CP or DP) promoted robust neuronal differentiation in wild-type EBs. Two-day-old wild-type EBs were treated with 25 μM of the indicated peptides and immunofluorescence analysis was performed on day 13 (n = ∼60 EBs/group, data are mean ± SD; **, *, p ≤ .03). The degree of neuronal differentiation was scored according to panel (C). (E): Gene expression analysis of mesoderm and neuronal markers. Two-day-old wild-type EBs were treated either with the indicated peptides at 25 μM (CP; BP) or vehicle (DMSO) and the expression profile of mesoderm/cardiac and neuronal markers was analyzed on day 13, by quantitative reverse transcription polymerase chain reaction. mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and presented as fold change in gene expression relative to the control (DMSO = 1). Data are mean ± SD, n ≥ 3; *, p < .05 **, p < .01. Abbreviations: AFP, alpha-fetoprotein; BP, blocking peptide; CP, control peptide; DAT, dopamine transporter; DMSO, dimethyl sulfoxide; DP, dimeric peptide; EB, embryoid body; ESC, embryonic stem cell; NFM, neurofilament medium polypeptide; TH, tyrosine hydroxylase. We previously showed that cripto−/− ESCs spontaneously differentiate into neurons during the time window when wild-type ESCs are primed for cardiomyogenesis by Cripto [12]. Thus, if specific and effective, the Cripto BP should be able to redirect ESC fate and to promote neural induction and neurogenesis in mESCs, even if they are cultivated in conditions (i.e., EBs in 15% FBS) that favor cardiac differentiation [34]. To evaluate the peptide(s) activity, 2-day-old wild-type EBs were treated with either CP, DP, or BP and both morphological and immunofluorescence analysis were performed on 13-day-old EBs (Fig. 3A). To better define the activity of the peptides, we used anti-βIII tubulin antibodies to identify neurons in culture and arbitrarily defined four grades of neuronal differentiation (Fig. 3C) ranging from the absence of neurons (Grade 0) to full neuronal differentiation (Grade 3), that is, presence of a dense network of βIII-tubulin-positive cells. The presence of either few isolated neurons or small areas of βIII-tubulin positive cells defined intermediate phenotypes, grade 1 and grade 2, respectively (Fig. 3C). Our data clearly indicated that Cripto BP but not DP or CP treatment promotes robust neuronal differentiation, characterized by a high percentage of grade 3 (full neuronal differentiation, Fig. 3C) phenotype (BP 45.6% ± 5.2 vs. CP 6.9% ± 0.6, Mean ± SD *, p ≤ .03; Fig. 3D). Accordingly, upon treatment with control peptides (CP or DP), the majority of the EBs scored showed a grade 0 (CP 75.9% ± 3.05 and DP 77.2% ± 7, vs. BP 17.4% ± 1.15, Mean ± SD, *, p ≤ .03; Fig. 3D). To support these findings, we performed the molecular analysis and examined the expression profile of neuronal differentiation markers in the same cardiac differentiation conditions, by quantitative reverse transcription polymerase chain reaction (QRT-PCR). In line with our previous findings in cripto−/− ESCs [12], expression of the pan neuronal marker NFM was significantly induced in 13-day-old EBs upon treatment with Cripto BP. Concomitantly, expression of the cardiac specific marker myosin light chain-2 ventricular as well as the mesendoderm marker alpha-fetoprotein were strongly reduced (Fig. 3E). Thus, our results suggest a fate switch in ESCs from mesoderm/cardiomyocytes to neurons. Accordingly, expression of the panmesodermal marker Brachyury/T was also strongly reduced in 2-day-old EBs upon treatment with Cripto BP (data not shown). Moreover, Cripto BP-treated wild-type EBs also showed induction of markers typical of DA development and differentiation such as, Pitx3 [38], Shh [39], Wnt5a [40], TH (the rate limiting enzyme in DA synthesis) and dopamine transporter (DAT; Fig. 3E), in line with previous findings in cripto−/− ESCs [12]. Thus, our results suggest that Cripto BP can enhance the neural differentiation of ES cells. Increased Dopaminergic Differentiation of ESCs Treated with Cripto BP We next examined whether Cripto BP treatment was able to further enhance DA differentiation of mESC, using the stromal cell-based protocol described by Barberi et al. [41], with minor modifications (Fig. 4A). In brief, mESCs were plated at low density onto stromal cells (PA6) for a period of 5 days, to promote neural induction. At day 5 the media was replenished and supplemented with Shh and FGF-8 (for ventral midbrain patterning). From day 8 to 11, cultures where changed into neuronal (N2) media in the presence of Shh and FGF-8 as well as basic FGF (to expand the pool of DA precursors). Finally (day 11–14), the cells were exposed to the survival factors brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF), and ascorbic acid. In all cultures, the CP, or Cripto BP was added to the differentiating ESCs from day 1 to day 11 (Fig. 4A). 4 Open in new tabDownload slide Cripto BP enhances neuronal induction and dopaminergic differentiation. (A): In vitro differentiation scheme. ESCs were seeded at low density onto a stromal feeder layer for 14 days in the presence of sequential morphogens and mitogens (Shh, FGF2, FGF-8) as well as the presence or absence of the CP or Cripto BP (12.5 μM). Differentiated cultures rarely contained pluripotent ESCs (Oct4, [B]), but contained numerous proliferating cells (Ki67+, [C]). No difference in Oct4+ or Ki67+ cells was detected between BP and CP, however BP cultures contained many more neural stem cells (rosettes, [D], and nestin+ cells, [E]) compared with CP (E′). Both CP and BP cultures contained ectodermal/epiderm cells (cytokeratin+ cells, [F]), however only CP cultures expressed mesodermal cells (CD29+ cells, [G]). The efficacy of neural induction in BP cultures was reflected by abundance of neural rosettes (D), and the dense neuronal and glial staining (Tuj+ and GFAP, respectively, [H]. (I): In vitro differentiation of ESCs in the presence of BP significantly increased the percentage of colonies containing neurons (TUJ1). (J): Cripto BP significantly increased the proportion of colonies containing TH+ dopaminergic neurons, and (K) the number of DA neurons/colony. (I–K): Mean ± SD, **, p < .01, ***, p < .0001. Photomicrographs illustrating TH+ neurons within neuronal-enriched ESC colonies following (L) CP administration, and (M–N) Cripto BP treatment. TH+ cells within the cultures acquired the expression of markers typical of midbrain DA neurons such as Nurr1 (O), Pitx3 (P), and GIRK2 (Q), confirming the DA phenotype of the TH+ neurons. Few cells with other neuronal phenotypes such as GABA (R) or 5HT (S) were present. No TH/GABA colocalization was detected. (T): Cripto BP-treated ESC cultures showed significantly increased dopamine and DOPAC release in vitro following KCl evoked release. Data represents mean ± SEM, n = 3, *, p < .05. Scale bar (B-H and L, M) = 200 μm, (N–S) = 50 μm. Abbreviations: 6HT, •••; AA, ascorbic acid; BDNF, brain-derived neurotrophic factor; BP, Cripto blocking peptide; CP, control peptide; DA, midbrain dopaminergic; DOPAC, dihydroxyphenylacetic acid; ESC, embryonic stem cell; FGF, fibroblast growth factor; GABA, γ-aminobutyric acid; GDNF, •••; GFAP, glial fibrillary acidic protein; PA6, •••; SRM, •••; TH, tyrosine hydroxylase. 4 Open in new tabDownload slide Cripto BP enhances neuronal induction and dopaminergic differentiation. (A): In vitro differentiation scheme. ESCs were seeded at low density onto a stromal feeder layer for 14 days in the presence of sequential morphogens and mitogens (Shh, FGF2, FGF-8) as well as the presence or absence of the CP or Cripto BP (12.5 μM). Differentiated cultures rarely contained pluripotent ESCs (Oct4, [B]), but contained numerous proliferating cells (Ki67+, [C]). No difference in Oct4+ or Ki67+ cells was detected between BP and CP, however BP cultures contained many more neural stem cells (rosettes, [D], and nestin+ cells, [E]) compared with CP (E′). Both CP and BP cultures contained ectodermal/epiderm cells (cytokeratin+ cells, [F]), however only CP cultures expressed mesodermal cells (CD29+ cells, [G]). The efficacy of neural induction in BP cultures was reflected by abundance of neural rosettes (D), and the dense neuronal and glial staining (Tuj+ and GFAP, respectively, [H]. (I): In vitro differentiation of ESCs in the presence of BP significantly increased the percentage of colonies containing neurons (TUJ1). (J): Cripto BP significantly increased the proportion of colonies containing TH+ dopaminergic neurons, and (K) the number of DA neurons/colony. (I–K): Mean ± SD, **, p < .01, ***, p < .0001. Photomicrographs illustrating TH+ neurons within neuronal-enriched ESC colonies following (L) CP administration, and (M–N) Cripto BP treatment. TH+ cells within the cultures acquired the expression of markers typical of midbrain DA neurons such as Nurr1 (O), Pitx3 (P), and GIRK2 (Q), confirming the DA phenotype of the TH+ neurons. Few cells with other neuronal phenotypes such as GABA (R) or 5HT (S) were present. No TH/GABA colocalization was detected. (T): Cripto BP-treated ESC cultures showed significantly increased dopamine and DOPAC release in vitro following KCl evoked release. Data represents mean ± SEM, n = 3, *, p < .05. Scale bar (B-H and L, M) = 200 μm, (N–S) = 50 μm. Abbreviations: 6HT, •••; AA, ascorbic acid; BDNF, brain-derived neurotrophic factor; BP, Cripto blocking peptide; CP, control peptide; DA, midbrain dopaminergic; DOPAC, dihydroxyphenylacetic acid; ESC, embryonic stem cell; FGF, fibroblast growth factor; GABA, γ-aminobutyric acid; GDNF, •••; GFAP, glial fibrillary acidic protein; PA6, •••; SRM, •••; TH, tyrosine hydroxylase. We first examined whether immature and proliferating cells remained in the cultures at the end of differentiation, day 14. Interestingly, Oct4+ cells (a pluripotent stem cell marker) as well as Ki67+ cells (a nuclear cell cycle marker) were present both in CP- and BP-treated cultures. However, while extremely few/rare Oct4+ cells were identified in CP (not shown) and BP (Fig. 4B), Ki67+ cells were relatively abundant both in CP (not shown) and BP cultures (Fig. 4C, BP). To further characterize the ESC cultures, we thus performed immunocytochemistry against nestin (neuroectoderm), cytokeratin (ectoderm/epidermis) and CD29 (mesoderm and ectoderm). We found an increase in the presence of neural rosettes and nestin staining in BP cultures (Fig. 4D, 4E) compared with CP cultures (Fig. 4E′). Although cytokeratin+ cells were detected in both CP- and BP-differentiated cultures (Fig. 4F, BP), few CD29+ cells were present only in CP cultures (Fig. 4G), suggesting that Cripto BP prevented mesoderm differentiation to promote neural induction, as described for cripto−/− ESCs [13, 14]. The efficacy of the neural induction was also evident by the abundance of both neurons (Tuj1) and glial cells (GFAP) in the BP cultures (Fig. 4H). We subsequently assessed the effect of BP on neuronal and DA differentiation in these culture conditions. First, we found that while treatment of cultures with CP had no effect on neural induction or TH differentiation, a clear increase was detected with BP (Fig. 4I–4K). Following differentiation in the presence of the Cripto BP, the majority of colonies within the cultures were immunoreactive for βIII-tubulin (91% compared with 66% in CP, Fig. 4I). Furthermore, Cripto BP was capable of significantly increasing the proportion of colonies containing TH-ir DA neurons (62%) compared with 34% in CP (Fig. 4J). Additionally, these TH-ir colonies contained significantly more DA neurons (TH+ cells/μm colony, 286%, Fig. 4K–4M). Interestingly, we found that DA differentiation of Cripto BP-treated ESCs could be further enhanced by addition of Noggin [28, 29] and Wnt5a [40, 42] (supporting information Fig. 6). Many of the TH-labeled neurons (in CP- and Cripto BP-treated cultures) showed a bipolar morphology (Fig. 4N). Immunohistochemistry for Nurr1, Pitx3, and G protein-activated inwardly rectifying potassium channel two (GIRK2; the latter, a marker preferentially expressed in SNpc DA neurons [43]), confirmed that TH-ir cells within the cultures (CP or BP treated) were of a midbrain DA phenotype (Fig. 4O–4Q) and, at least a notable proportion possessed a SNpc phenotype (Fig. 4Q). Interestingly, few cells within the cultures were immunoreactive for other neuronal subpopulations markers including γ-aminobutyric acid (GABA-ir) and serotonin (5HT-ir) (Fig. 4R–4S). Furthermore, we analyzed the ability of the dopamine cells generated in vitro to synthesize, release and utilize dopamine. Although both Cripto BP and CP-treated cultures were able to utilize DA (as reflected by dopamine turnover; ratio of DOPAC to DA, Fig. 4T′′′), we noted that Cripto BP treatment of mESCs significantly increases the KCl-evoked release of dopamine (Fig. 4T′) and DOPAC (Fig. 4T″) within the cultures, reflective of enhanced DA differentiation. Thus, our results show that Cripto BP, in addition to promoting neural induction (as shown in EB cultures, Fig. 3), was also capable of enhancing the number of DA neurons by 2.8-folds in a neural media (Fig. 4). Enhanced Functional Recovery in Parkinsonian Rats Receiving ESC Grafts Treated with Cripto BP We next examined the ability of mESC-derived DA neurons to survive, integrate and induce functional recovery in an animal model of PD. Previous studies on the function of cripto in the DA differentiation of ESCs in vivo have been performed using cripto−/− ESCs grown for 4 days as EBs [13, 14]. Given the high yield of DA neurons in wt mESCs treated with Cripto BP, cells differentiated as described in supporting information Figure 6, in the presence of either Cripto BP or CP, were used for transplantation in 6-hydroxydopamine lesioned rats, an animal model of PD. The validity of the model was assessed by amphetamine-induced rotational behavior and histological examination, which confirmed that all animals had near complete unilateral lesions of SNpc DA neurons (cell loss >90% and >6 rotations/minute; data not shown). When the viability of the grafts was examined, we found that 97% of the animals (31 of 32 rats) transplanted with 12,000 mESC-derived cells showed viable grafts at 8 weeks. The majority of the rats receiving ESCs pretreated with CP showed the presence of tumors/overgrowths within the grafts (Fig. 5A, 5B). Interestingly, tumor formation was reduced by more than half, but not abolished, in rats receiving ESCs pretreated with Cripto BP (Fig. 5A, 5C). To determine the nature of these tumors (i.e., neural overgrowths or teratomas), we examined the grafts by immunohistochemistry and scored the cell types (Fig. 5E–5K, see “Methods” section). The majority of these tumors (all CP-pretreated and 3/4 of BP-pretreated ESCs) contained ectodermal-epidermal and mesodermal cells, indicating that they were teratomas. This was confirmed by the presence of scarce Oct4+ cells in these grafts (Fig. 5K). Thus, while mesodermal cells were not observed after Cripto BP treatment in vitro, these cells appeared in vivo, indicating that the level of antagonism may not be sufficient to prevent mesoderm formation from the few remaining ESCs in vivo [14]. 5 Open in new tabDownload slide Cripto BP reduces the incidence and size of tumors following mouse embryonic stem cells transplantation. (A): Pretreatment of embryonic stem cells (ESCs) with BP reduced tumor incidence upon transplantation compared with CP treatment. Neural red-stained sections showed that upon tumor occurrence, animals receiving Cripto BP-pretreated ESCs (C) showed significantly reduced tumor volume by comparison to CP treatment (B). (D): Semiquantitative analysis of graft components within tumors (refer to “Material and Methods” section for scoring). (E): Tumors contained abundant proliferating cells (Ki67+; [E, G]) and undifferentiated neural cells (Nestin+; [F, G]) within the graft. However, in all cases, these tumors also contained cells from other germ layer cells including mesoderm (CD29+, [H]), endothelial cells (CD29+, [I]), and ectoderm/epiderm cells (cytokeratin+, [J]) as well as the rare presence of undifferentiated ESCs (Oct4+, [K]), indicating that the tumors were teratomas. Student's t-test, * p < .05. Abbreviations: BP, Cripto blocking peptide; CP, control peptide; CPu, caudate putamen; Ctx, cortex; Lv, lateral ventricle. 5 Open in new tabDownload slide Cripto BP reduces the incidence and size of tumors following mouse embryonic stem cells transplantation. (A): Pretreatment of embryonic stem cells (ESCs) with BP reduced tumor incidence upon transplantation compared with CP treatment. Neural red-stained sections showed that upon tumor occurrence, animals receiving Cripto BP-pretreated ESCs (C) showed significantly reduced tumor volume by comparison to CP treatment (B). (D): Semiquantitative analysis of graft components within tumors (refer to “Material and Methods” section for scoring). (E): Tumors contained abundant proliferating cells (Ki67+; [E, G]) and undifferentiated neural cells (Nestin+; [F, G]) within the graft. However, in all cases, these tumors also contained cells from other germ layer cells including mesoderm (CD29+, [H]), endothelial cells (CD29+, [I]), and ectoderm/epiderm cells (cytokeratin+, [J]) as well as the rare presence of undifferentiated ESCs (Oct4+, [K]), indicating that the tumors were teratomas. Student's t-test, * p < .05. Abbreviations: BP, Cripto blocking peptide; CP, control peptide; CPu, caudate putamen; Ctx, cortex; Lv, lateral ventricle. Interestingly, despite more nestin+ cells were detected in BP compared with CP-pretreated ES cells (Fig. 5D), we did not detect areas with very high cell density/parenchymal distortion containing nestin+ cells and Ki67+ cells in the absence of mesodermal and endodermal cells, indicating that there were no cases of isolated neural overgrowth (data not shown). Remarkably, in the cases where tumors were observed, animals receiving ESCs pretreated with Cripto BP showed a significant reduction in tumor volume (0.94 × 1010 μm3 ± 0.27 × 1010) compared with CP (2.22 × 1010 μm3 ± 0. 44 × 1010, Mean ± SEM, *, p < .05), as assessed in neural red-stained sections (Fig. 5B–5C). Thus, our results indicate that Cripto BP reduces the incidence and size of mESC-derived teratomas by ∼60%. We next examined the impact of ESC transplantation on amphetamine-induced circling behavior and dopamine neuron engraftment in rats that did not present any tumor (n = 11 Cripto BP grafts and n = 4 CP grafts, 15 BP grafts and 16 CP grafts of the total). Functionally, the motor behavior of parkinsonian rats receiving ESCs grafts significantly improved by 8 weeks compared with sham-treated animals (Fig. 6A). Grafting of 12,000 ESCs pretreated with CP showed modest levels of functional improvement compared with shams from 4 to 8 weeks. Most remarkably, animals receiving the same number of ESCs treated with the Cripto BP showed significant reductions in amphetamine-induced rotational behavior as early as week 2 postgrafting. Moreover, the reduction in motor asymmetry progressively (and significantly) improved from 6 weeks postgrafting compared with CP grafts (Fig. 6A). 6 Open in new tabDownload slide Cripto BP induces behavioral and anatomical improvements in embryonic stem cells (ESC)-grafted parkinsonian rats. (A): Treatment of ESCs with Cripto BP (red line) significantly improves amphetamine-induced rotational behavior of grafted animals compared with CP-treated grafts (blue line) and sham grafts (green). (B): An increase in the number of TH-ir cells/μm3 was detected within the graft following transplantation of ESCs treated with Cripto BP compared with CP. (C): Correlation of TH+ cells and functional improvement in grafted animals. No statistical difference was detected between CP (blue) and Cripto BP (red). Bold line represents regressions and faint lines, 95% confidence intervals. (D): Photomicrographs illustrating TH-ir neurons within the striatum following grafting of ESCs treated with CP and (E) Cripto BP. (F): Numerous hypertrophied TH+ fibers seen outside the graft site, indicative of good graft integration. (G): High power image of a TH cell within the graft showing the bipolar morphology. (H): TH+/Nurr1+, (I) TH+/DAT+, (J) TH+/Pitx3+, (K) TH+/GIRK2+ (fill arrowhead) staining within the graft, confirming the midbrain dopaminergic phenotype of the TH neurons in Cripto BP grafts. (L): Norepinephrine (labeled with NET) fibers could be seen throughout the graft but did not colocalize with TH. TH neurons within the grafts did not colocalize with (M) GABA or, serotonin (5HT (5-hydroxitriptamine), not shown). Data are mean ± SD. *, p < .05, **, p < .01, ***, p < .001. Scale bar (Di, Ei) = 400 μm, (Dii, Diii, Eii, G) = 100 μm, (F, H, I–M) = 50 μm. Abbreviations: BP, Cripto blocking peptide; CP, control peptide; DAT, dopamine trasporter; GABA, γ-aminobutyric acid; NET, norepinephrine transporter; TH, tyrosine hydroxylase; GIRK2, G protein-activated inwardly rectifying potassium channel two; Nurr1, nuclear receptor related one; Pitx3, paired-like homeodomain transcription factor three. 6 Open in new tabDownload slide Cripto BP induces behavioral and anatomical improvements in embryonic stem cells (ESC)-grafted parkinsonian rats. (A): Treatment of ESCs with Cripto BP (red line) significantly improves amphetamine-induced rotational behavior of grafted animals compared with CP-treated grafts (blue line) and sham grafts (green). (B): An increase in the number of TH-ir cells/μm3 was detected within the graft following transplantation of ESCs treated with Cripto BP compared with CP. (C): Correlation of TH+ cells and functional improvement in grafted animals. No statistical difference was detected between CP (blue) and Cripto BP (red). Bold line represents regressions and faint lines, 95% confidence intervals. (D): Photomicrographs illustrating TH-ir neurons within the striatum following grafting of ESCs treated with CP and (E) Cripto BP. (F): Numerous hypertrophied TH+ fibers seen outside the graft site, indicative of good graft integration. (G): High power image of a TH cell within the graft showing the bipolar morphology. (H): TH+/Nurr1+, (I) TH+/DAT+, (J) TH+/Pitx3+, (K) TH+/GIRK2+ (fill arrowhead) staining within the graft, confirming the midbrain dopaminergic phenotype of the TH neurons in Cripto BP grafts. (L): Norepinephrine (labeled with NET) fibers could be seen throughout the graft but did not colocalize with TH. TH neurons within the grafts did not colocalize with (M) GABA or, serotonin (5HT (5-hydroxitriptamine), not shown). Data are mean ± SD. *, p < .05, **, p < .01, ***, p < .001. Scale bar (Di, Ei) = 400 μm, (Dii, Diii, Eii, G) = 100 μm, (F, H, I–M) = 50 μm. Abbreviations: BP, Cripto blocking peptide; CP, control peptide; DAT, dopamine trasporter; GABA, γ-aminobutyric acid; NET, norepinephrine transporter; TH, tyrosine hydroxylase; GIRK2, G protein-activated inwardly rectifying potassium channel two; Nurr1, nuclear receptor related one; Pitx3, paired-like homeodomain transcription factor three. Subsequently, we examined whether the functional improvement observed in Cripto BP-treated animals was related to an increase in TH-ir neuron counts within the grafts of tumor-free animals. Importantly, the volume of the grafts was not significantly different between treatments (CP = 8.79 × 108 ± 1.20 × 108 and BP = 9.43 × 108 ± 0.88 × 108, p = .687), and no TH neurons were found within the striatum of animals receiving sham grafts (data not shown). When we examined the number of TH cells, we found that animals grafted with Cripto BP-treated ESCs showed a consistent increase in TH+ cells/μm3 compared with CP-treated ESCs (p = .046, Fig. 6B–6E). However, we found no statistically significant difference between CP and BP-pretreated ESC grafts when examining the correlation between behavioral improvement and TH+ neuron number (Fig. 6C), indicating that the improvement is mainly contributed by an increase in TH+ neurons. We observed that many of the TH+ cells within the grafts displayed a bipolar morphology (Fig. 6G) as well as an extensive network of hypertrophied neurites throughout the striatum (Fig. 6F). Immunohistochemistry revealed that many of the TH-ir cells within the grafts also coexpressed Nurr1, DAT, Pitx3, and GIRK2, verifying their midbrain DA identity (Fig. 6H–6K). Interestingly, Pitx3/TH and GIRK2/TH colabeling illustrated that several TH+ neurons within the grafts were SNpc midbrain-like DA neurons, both in CP and BP grafts, (filled arrowheads in Fig. 6K). Although norepinephrine fibers (labeled with norepinephrine transporter, NET) could be seen within the grafts (Fig. 6L), no TH-ir cell bodies within the graft colocalized with other neurotransmitters, including norepinephrine (Fig. 6L), GABA (Fig. 6J), and serotonin (not shown). In summary, our results illustrate that a synthetic small peptide, Cripto BP, improves the generation of midbrain DA neurons from ESCs in vitro and in vivo, reduces the incidence of teratomas and results in a behavioral improvement in parkinsonian rats. Discussion In this study, we report the identification (by a combinatorial screening) of a novel small synthetic peptide (Cripto BP) that antagonizes the onco-developmental signaling molecule, Cripto [2]. Although Cripto blocking antibodies have been used in the past to suppress tumor cell growth [44], synthetic molecules/peptides offer numerous advantages over biotherapeutics as they are easier and cheaper to produce, more stable, and generally free of contaminants of biological origin. Combinatorial screenings have been successfully used in the last two decades to identify small molecules/peptides for use in drug discovery and cell biology [45]. Recently, cell-based screenings have identified synthetic molecules that regulate stem cell differentiation/proliferation, which have been used as probes to study the underlying biology of stem cells [46–49]. Although many basic questions regarding identification of targets and mechanisms of action of the molecules identified in cell-based screenings still remain to be answered, it has become evident that such synthetic molecules could lead to significant advances in stem cell biology. In our study we used a reverse screening approach using a defined mechanism/target, the Cripto/ALK-4 receptor complex, and therefore screened a synthetic peptide library to identify a Cripto BP. The choice of a synthetic peptide library was made as peptides are known to be optimal probes to target the extracellular domains of membrane receptors, provided their typical high affinity and selectivity, minimal drug-drug interactions, low accumulation capacity, and low toxicity [31]. Moreover, tetrameric peptides are extremely flexible molecules, have a high-recognition surface, are highly resistant to proteases and can thus be applied in functional assays and in a broad range of applications both in vitro and in vivo. Based on these considerations, we designed an assay to identify competitor peptides of the Cripto/ALK-4 receptor complex that could be used to control ESC fate. Consistent with this idea, we found that Cripto BP prevented Cripto-dependent Smad2 phosphorylation, blocked cardiomyogenesis and favored neurogenesis in ESCs, mimicking the phenotype of cripto−/− ESCs [12, 13]. Thus, these data and our in vitro results showing that Cripto BP selectively binds Cripto and prevent its in vitro binding to ALK-4 receptor, support the idea that, despite the function of ALK-4 as a receptor for multiple TGFβ-related ligands [50], the neutralizing activities that we detect are the result of specific targeting of the Cripto protein. Interestingly, the BMPs antagonist Noggin was previously shown to induce neuroectodermal cell development and enhance production of DA neurons from hESCs [28, 29]. Moreover, it has been recently reported that a combination of Noggin and the small molecule SB431542, which inhibits ALK-4, -5, and -7 receptors [51] and blocks SMAD signaling, efficiently induce neural conversion of human pluripotent stem cells and allow their subsequent DA differentiation [30]. Our protocol differs from previous studies in that we developed and used a selective inhibitor (Cripto BP), instead of broader inhibitors such as Noggin and particularly SB431542, which blocks signaling by several TGF-β superfamily ligands. Our work indicates that a selective blocking with Cripto BP is sufficient to improve neural differentiation of mESCs in vitro. This improvement was not at the expense of an increased differentiation of Oct4+ cells, as their number did not change by Cripto BP, but was rather due to a switch from mesodermal to neural differentiation. We thus suggest that additional cell sorting or BMP inhibitors could further improve this protocol. In agreement with this, we found that Noggin and Wnt5a enhanced the production of DA neurons by Cripto BP in vitro. These findings indicate that Cripto BP can indeed be used in combination with other factors to further improve DA differentiation. With regard to cell transplantation in animal models of PD, Chiba et al. [29], previously found that grafting of hemiparkinsonian rats with hESCs-derived DA neurons pretreated with Noggin lead to significant engraftment of DA neurons and behavioral improvement, but all animals showed cell overgrowths. These results differ from the study by Sonntag et al. [28], who showed teratomas in about 25% of the animals grafted with Noggin-treated cells and behavioral improvement that correlated with TH+ cell numbers in only two of the grafted animals. In our study, we found that Cripto BP treatment, led to grafts containing many more TH+ cells (9,304 ± 850 for BP and 5,841 ± 1,939 for CP), and to a clear behavioral improvement in hemiparkinsonian rats. However, our approach did not entirely eliminate tumors. Cripto BP significantly reduced the incidence of teratoma formation (by 58%) and the size of tumors, but teratomas were still observed in as many as 27% of the grafted rats, even in the presence of additional factors, that is, Wnt5a and Noggin. Previous studies have shown that while transplantation of as few as 20,00 cripto−/− ES-derived EB cells resulted in functional recovery and no tumor/overgrowth formation ESCs [13], 50,000 cripto−/− ES-derived EB cells did not prevent tumor/overgrowth formation, but lead to smaller grafts that contained more neurons [14]. We thus explored whether different cell grafting densities of mESCs, differentiated as described in Figure 4A, could reduce tumor/outgrowth formation while maintaining behavioral improvement, but found that cell density plays a seemingly insignificant role in predifferentiated mESCs in the presence of Cripto BP (supporting information Fig. 7). We also explored whether the efficiency of Cripto blocking with Cripto BP may have decreased in vivo, but found that infusion of Cripto BP peptide in vivo did not further enhance the effect of Cripto BP pretreatment in vitro (data not shown). Thus, our results suggest that additional factors present during DA differentiation and/or after grafting may play an additional role in tumor/outgrowth formation. These data are thus in agreement with the results by Sonntag et al., who suggested that activation of other pathways, in a cell autonomous or nonautonomous manner, may also contribute to cell overgrowth in vivo [14]. Finally, it should be noted that few previous studies have reported robust engraftment of mES cell-derived DA neurons in animal models of PD in the absence of tumor formation by either using transgenes [52, 53], feeders [41] or knockout ES cells [13] (see [20] for review). Our differentiation protocol, while aiming at avoiding genetic modification, requires the use of PA6 feeders. In our protocol, we introduced some modifications to the protocol by Barberi, which together with some variability introduced by PA6 cocultivation, may have contributed to incomplete specification of the ESCs and tumor formation. In the future, it will thus be important to determine whether additional instructive/differentiation factors, inhibitors of pathways involved in tumor formation and cell separation techniques, can be used to improve the functional integration of ES-derived DA neurons in models of PD, making feeder-free and transgene-free DA differentiation protocols safer, as well as more reproducible and robust. Conclusion In conclusion, our strategy provides evidence that a small synthetic peptide obtained from an in vitro screen can not only target specific proteins and signaling pathways in vitro, but can also enhance neural induction in mESCs, midbrain DA neuron differentiation and DA release. More importantly, Cripto BP can improve the number and the functional integration of ESC-derived DA neurons, leading to behavioral improvements in animal models of PD. This is the first time that a reverse screening approach has been used in stem cell biology to improve stem cell transplantation. However, the presence of teratomas in a significant number of the grafted animals remains as an important limiting factor. Hence, further efforts are required to elucidate the mechanisms responsible for tumor formation and optimize CRT. Future studies will examine whether hESCs respond to Cripto BP in a similar manner as mESCs and whether the screening and targeting of additional signaling pathways may improve the therapeutic application of stem cells in CRT for PD. Acknowledgements We are grateful to the members of the Stem Cell Fate Laboratory and the Arenas laboratory for helpful discussion. This work was supported by the Telethon Foundation to G.M. (grants GGP05112, GGP08120) and S.D.F. (GGP08062), Associazione Italiana Ricerca sul Cancro (AIRC) to G.M. and S.D.F.; and by grants from the European Union (Eurostemcell), the Swedish Research Council (VR2008), Norwegian Research Council and Karolinska Institutet, to E.A. M.R. is funded by a FIRB (project, n° RBNE03PX83_005). C.L.P. was supported by a Human Frontiers Science Program Long-term Fellowship and a National Health and Medical Research Council, Australia Career Development Award. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Authors contributions: E.L.: conception and design, provision of study material, collection and/or assembly of data, data analysis and interpretation, manuscript writing; C.L.P.: conception and design, provision of study material, collection and/or assembly of data, data analysis and interpretation, manuscript writing; S.P.: collection and/or assembly of data, data analysis and interpretation; D.M.: collection and/or assembly of data, data analysis and interpretation; D.R.: collection and/or assembly of data; M.R.: conception and design, collection and/or assembly of data, provision of study material, data analysis and interpretation; S.D.F.: conception and design, collection and/or assembly of data, data analysis and interpretation; E.A.: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing; G.M.: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing. E.L, C.L.P. equally contributed as first author. E.A and G.M. equally contributed as last author. Disclosure of potential conflicts of interest is found at the end of this article. First published online in STEM CELLS EXPRESS July 16, 2010. Telephone: 46-8-5248-7663; Fax: 46-8-34-1960 Telephone: 39-081-6132357; Fax: 39-081-6132595 Copyright © 2010 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Small Synthetic Cripto Blocking Peptide Improves Neural Induction, Dopaminergic Differentiation, and Functional Integration of Mouse Embryonic Stem Cells in a Rat Model of Parkinson's Disease  
JF - Stem Cells
DO - 10.1002/stem.458
DA - 2010-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-small-synthetic-cripto-blocking-peptide-improves-neural-induction-Cd0o0q3PF5
SP - 1326
EP - 1337
VL - 28
IS - 8
DP - DeepDyve
ER -