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TISSUE-SPECIFIC STEM CELLS Inhibition of Notch Signaling in Human Embryonic Stem Cell–Derived Neural Stem Cells Delays G1/S Phase Transition and Accelerates Neuronal Differentiation In Vitro and In Vivo LODOVICA BORGHESE,a DASA DOLEZALOVA,b,c THORALF OPITZ,a SIMONE HAUPT,a ANKE LEINHAAS,a BARBARA STEINFARZ,a PHILIPP KOCH,a FRANK EDENHOFER,a ALES HAMPL,b,c OLIVER BRÜSTLEa a Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Bonn, Germany; bBiology Department, Faculty of Medicine, Masaryk University, Brno, Czech Republic; c Department of Molecular Embryology, Institute of Experimental Medicine, ASCR, v.v.i., Prague, Czech Republic Key Words. Neural stem cells • Notch • Neuron • Cell cycle ABSTRACT The controlled in vitro differentiation of human embryonic stem cells (hESCs) and other pluripotent stem cells provides interesting prospects for generating large numbers of human neurons for a variety of biomedical applications. A major bottleneck associated with this approach is the long time required for hESC-derived neural cells to give rise to mature neuronal progeny. In the developing vertebrate nervous system, Notch signaling represents a key regulator of neural stem cell (NSC) maintenance. Here, we set out to explore whether this signaling pathway can be exploited to modulate the differentiation of hESCderived NSCs (hESNSCs). We assessed the expression of Notch pathway components in hESNSCs and demonstrate that Notch signaling is active under self-renewing culture conditions. Inhibition of Notch activity by the c-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenyl- glycine t-butyl ester (DAPT) in hESNSCs affects the expression of human homologues of known targets of Notch and of several cell cycle regulators. Furthermore, DAPT-mediated Notch inhibition delays G1/S-phase transition and commits hESNSCs to neurogenesis. Combined with growth factor withdrawal, inhibition of Notch signaling results in a marked acceleration of differentiation, thereby shortening the time required for the generation of electrophysiologically active hESNSC-derived neurons. This effect can be exploited for neural cell transplantation, where transient Notch inhibition before grafting suffices to promote the onset of neuronal differentiation of hESNSCs in the host tissue. Thus, interference with Notch signaling provides a tool for controlling human NSC differentiation both in vitro and in vivo. STEM CELLS 2010;28:955–964 Disclosure of potential conflicts of interest is found at the end of this article. INTRODUCTION Pluripotency and unlimited self-renewal make human embryonic stem cells (hESCs) an attractive source for generating large numbers of somatic cells in vitro. This is particularly relevant for cells from tissues with low regenerative potential such as heart, insulin-producing cells and neurons. Different protocols for the derivation of neural cell types from hESCs have been published [1, 2]. Our group has established a homogeneous population of long-term self-renewing neural stem cells (NSCs) from hESCs (lt-hESNSCs) that are propagated continuously in the presence of fibroblast growth factor-2 (FGF2)/epidermal growth factor (EGF) [3]. On FGF2/EGF withdrawal, lt-hESNSCs differentiate into neurons and, at later time points, also into glial cells. Lt-hESNSCs represent a readily accessible system to model human neural lineage development and may serve as a renewable cell source for pharmaceutical and medical applications. A major limitation in the biomedical use of hESCderived neural cells is that they require, in contrast to cells from other vertebrates, extensive time to differentiate into specific functional subtypes. It takes several weeks for a human neuron to differentiate from a naı̈ve stem cell and become physiologically mature [3, 4]. Studies aimed at overcoming this limitation should address the mechanisms Author contributions: L.B.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; D.D.: collection and assembly of data, data analysis and interpretation; T.O.: collection and assembly of data, data analysis and interpretation; S.H.: collection and assembly of data, data analysis and interpretation; A.L.: support with the establishment of in vivo experiments; B.S.: support with the establishment of rat hippocampus slice cultures; P.K.: provision of study material; F.E.: provision of study material, manuscript writing; A.H.: data interpretation, manuscript writing; O.B.: conception and design, data interpretation, manuscript writing, final approval of manuscript. Correspondence: Oliver Brüstle, M.D., Institute of Reconstructive Neurobiology, University of Bonn, LIFE & BRAIN Center, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. Telephone: þ49-228-6885-500; Fax: þ49-228-6885-501; e-mail: brustle@uni-bonn. de Received October 14, 2009; accepted for publication March 3, 2010; first published online in STEM CELLS EXPRESS March 16, 2010. C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.408 V STEM CELLS 2010;28:955–964 www.StemCells.com Notch Inhibition in Human Neural Stem Cells 956 responsible for the balance between stem cell proliferation and differentiation. Notch is a key regulator of NSC maintenance in the developing nervous system. Data generated in different vertebrates showed that Notch is required to maintain neural precursors in an undifferentiated self-renewing state, while preventing premature neurogenesis [5, 6]. Notch signaling is dependent on cell–cell interaction. Binding of the Notch receptor to a ligand of the Delta/Serrate/Lag2 family on an adjacent cell triggers a series of proteolytic cleavages, the last being mediated by the c-secretase complex. As a result, the Notch intracellular domain is released and enters the nucleus, where it associates with the DNA-binding protein CSL (CBF1/RBPjk/Su(H)/Lag1) to assemble a transcriptional complex that activates downstream targets [7]. In the context of NSCs, Notch activation leads to the transcription of Hairy and enhancer of split (Hes) and Hes-related with YRPW motif (Hey) basic helix-loop-helix (bHLH) repressor genes, which in turn antagonize proneural genes such as Mash1 and neurogenins [5, 8]. In this work, we assessed which members of the Notch pathway are expressed in lt-hESNSCs, and whether Notch signaling is required for the maintenance of lt-hESNSCs. Using the c-secretase inhibitor (GSI) DAPT to interfere with Notch activity, we show that Notch plays an essential role in maintaining lt-hESNSC self-renewal, by contributing to progression through G1/S-phase of the cell cycle and preventing premature neurogenesis. Inhibition of Notch signaling also largely enhances neuronal differentiation induced by FGF2/ EGF withdrawal, thereby accelerating the generation of lt-hESNSC-derived neurons in vitro and in vivo. MATERIALS AND METHODS Culture of Lt-hESNSCs Lt-hESNSCs (H9.2 and I3 lines) were cultured on polyornithin/ laminin (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich. com) coated dishes as previously described [3]. Differentiation was performed on matrigel (BD Biosciences, San Diego, CA, http:// www.bdbiosciences.com) [3]. DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich), was used at a final concentration of 2.5 lM. DMSO (0.1%) was used as vehicle control. Electrophysiological Analysis For patch-clamp measurements cells were superfused with aCSF at 1–2 ml/min, which contained (in millimolars): 140 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 25 D-glucose, and 10 Hepes/ NaOH (pH 7.35, 305–315 mOsmol/kg). Whole cell currentclamp and voltage-clamp recordings were performed at room temperature (rt) with an Axopatch-200B amplifier (Axon Instruments, Union City, CA, http://www.axon.com) interfaced by an A/D-converter (Digidata 1320, Axon) to a PC running PClamp software (Version 9, Axon). For whole cell voltage-clamp recordings, the patch pipette (tip resistance 3–5 MX) contained (in millimolars): 120 potassium gluconate (C6H11O7K), 20 KCl, 10 NaCl, 10 EGTA, 1 CaCl2, 4 Mg ATP, and 0.4 Na GTP, 10 HEPES/KOH (pH 7.2, 280–290 mOsmol/kg). Whole cell currents were low-pass filtered at 2 kHz and recorded at a rate of 10 kHz. Membrane potential was recorded at 50 kHz whereas the low-pass filter was set to 10 kHz. Organotypic Slice Cultures Four hundred micrometer thick slices encompassing the dentate gyrus, hippocampus, and entorhinal/temporal cortex were prepared from 9-day-old Wistar rats (Charles River, Sulzfeld, Germany, http://www.criver.com), using a vibroslicer (VSLM1, http://www. campdeninstruments.com), and propagated as interface cultures on clear polyester membranes (Transwell-Clear, Corning, Amsterdam, Netherlands, http://www.corning.com/lifesciences) [9]. After 3-day in vitro differentiation, 15 104 lt-hESNSCs (I3) expressing green fluorescent protein (GFP) were resuspended with Alfazyme (PAA Laboratories, Cölbe, Germany, http://www.paa.com) and plated onto the dentate gyrus of 1-week-old organotypic slices. Grafted slices were cultured for 4 weeks. Cell Transplantation 9- to 12-week-old severe combined immunodeficiency (SCID)beige mice (Jackson Laboratory, Bar Harbor, ME, http:// www.jax.org) were anesthetized and injected intracranially in the mid-striatum with 105 GFP-expressing lt-hESNSCs (I3). Two and four weeks after transplantation, animals were deeply anesthetized and perfused with 4% paraformaldehyde (PFA). Brains were prepared for frozen sectioning, mounted in Tissue-Tek (Sakura, Zoeterwoude, Netherlands, http://www.sakuraeu.com), and cut into 30-lm thick slices using a cryostat. Animal care was in accordance with institutional guidelines. Reverse Transcription-Polymerase Chain Reaction RNA was extracted using peqGOLD TriFast (peqlab, Erlangen, Germany, http://www.peqlab.de), and DNaseI-treated (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). cDNA was generated with iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, http://www. bio-rad.com). Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using Taq Polymerase (Invitrogen) with omission of reverse transcriptase or cDNA serving as negative controls. cDNA from RNA extracts of human fetal or adult brain (Stratagene, La Jolla, CA, http://www.stratagene.com) were used as positive controls. Quantitative real-time RT-PCR (qRT-PCR) analyses were performed in triplicates on a BioradiCycler using SYBRI-green detection method. PCR products were assessed by dissociation curve and gel electrophoresis. Data were normalized to 18S rRNA levels and analyzed using the DDCt method. Primers used are given in supporting information Table 1. Western Blotting For analysis of cleaved NOTCH1, lt-hESNSCs (I3) were resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% IGEPALV CA-630 in ddH2O, Proteinase Inhibitor Cocktail Set III [1:100, Calbiochem, San Diego, CA, http://www.merckchemicals.com]), and protein extracts loaded onto a 10% SDSPAGE gel followed by transfer onto nitrocellulose membrane (Carl-Roth, Karlsruhe, Germany, http://www.carl-roth.de). SDS lysis buffer 1% (50 mM Tris-HCl, pH 6.8; 1% SDS; 10% glycerol), sonication and polyvinylidene fluoride membranes were used for analysis of cell cycle regulators. For antibodies, see supporting information Table 2. R Immunochemistry Cells were fixed in 4% PFA, permeabilized with 0.1% Triton X-100, and stained over/night (o/n) at 4 C with primary antibodies. Organotypic rat slice cultures, fixed in PFA 4% o/n at 4 C, and cryoslices from mouse brains were permeabilized with 0.1%– 0.3% Triton X-100, and stained o/n at rt with primary antibodies. Samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). For the quantification in Figures 1 and 3 over 500 cells were assessed in each experiment. Quantitative analysis in Figure 5G was performed by comparing values of fluorescence intensity/area in the graft relative to the surrounding host tissue (as in [10]) in three independent transplants. Antibodies used are listed in supporting information Table 2. Bromodeoxyuridine and Cell Cycle Analysis Lt-hESNSCs (I3) were incubated with bromodeoxyuridine (BrdU, 10 lM f.c., Sigma-Aldrich) for 3.5 hours at 37 C, fixed in 4% Borghese, Dolezalova, Opitz et al. 957 Figure 1. Notch signaling is active in lt-hESNSCs and its inhibition by DAPT results in reduction of BrdU incorporation and promotion of neuronal differentiation. (A): Western blot analysis showing actively cleaved NOTCH1-ICD in lt-hESNSCs (NICD, NSCs lane). Note the absence of NICD on treatment with the c-secretase inhibitor DAPT (þDAPT). DMSO is used as vehicle control. Beta-actin was used as protein loading control. (B): Semiquantitative RT-PCR analysis reveals differences in transcript levels for direct and indirect target genes of Notch between lt-hESNSCs (I3) treated with DAPT (þ) and their untreated counterparts (–). 18S rRNA levels were used as quantitative reference. (C): Quantitative real-time RTPCR analysis of known direct and indirect Notch target genes for lt-hESNSCs (I3) treated with DMSO or DAPT, in comparison with untreated lthESNSCs (–) (baseline sample, equal to 1). Data, normalized to reference 18S rRNA levels, are presented as mean þ SEM (n ¼ 3). *, p < .05. (D– F): Immunofluorescence analysis of lt-hESNSCs after 4 days under self-renewing culture conditions (untreated), in the presence of DAPT (þDAPT), and in the presence of vehicle (þDMSO), stained with antibodies to b-III tubulin (D–F) and BrdU (D0 –F0 ). DAPI labels nuclei (D00 –F00 ). Scale bars ¼ 50 lm. (G): Histograms showing the percentage of b-III tubulin-positive cells in the three different conditions depicted in D–F. Data are presented as mean þ SEM (n 4). *, p .01. (H): Histograms showing the percentage of BrdU-positive cells for the samples in D0 –F0 , normalized to the total number of cells minus the number of cells positive for b-III tubulin. Data are presented as mean þ SEM (n 4). **, p < .05. Abbreviations: BLBP, brain lipid-binding protein; BrdU, bromodeoxyuridine; DAPI, 40 ,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]S-phenylglycine t-butyl ester; DMSO, dimethylsulfoxide; lt-hESNSCs, long-term self-renewing human embryonic stem cell–derived neural stem cells; M, molecular weight marker; NICD, intracellular domain of NOTCH1; NSCs, neural stem cells. PFA, permeabilized with 0.5% Triton X-100, treated with 2 N HCl and subsequently with 0.1 M borate buffer, and stained o/n with primary antibody. For cell cycle analysis, cells incubated with BrdU were resuspended in PBS containing Hoechst 33342 (1.2 lg/ml, Invitrogen), and processed using the FITC BrdU Flow Kit (BD Biosciences). Flow cytometry was performed on a LSRII (BD Biosciences); data analysis was done using the FlowJo software (Tree Star, Ashland, OR, http://www.treestar. com). www.StemCells.com RESULTS Notch Pathway Components Are Expressed and Notch Signaling Is Active in Self-Renewing Lt-hESNSCs Notch signaling is known to contribute to NSC maintenance during neural development. To understand whether main- 958 tenance of lt-hESNSCs depends also on Notch signaling, we first assessed the expression of Notch pathway components by qRT-PCR. We compared transcript levels in lt-hESNSCs derived from two hESC lines (I3 and H9.2) and human fetal brain, using human adult brain as reference point. Members of the c-secretase complex (Anterior Pharynx-defective 1 homolog A, APH1A and Presenilin 1, PSEN1), the human homologues of Notch (NOTCH1, NOTCH2, and NOTCH3), the human homologues of Delta and Serrate ligands (Deltalike DLL1, DLL3, DLL4, and JAGGED1, with the exception of JAGGED2), and the human homologues of bHLH repressors (HES1, HES5, and HEY1) were expressed in both H9.2 and I3 lt-hESNSCs at similar levels (supporting information Fig. 1A). The proneural gene ASCL1 (homologue of Mash1), a known target of the Hes/Hey gene-mediated transcriptional repression, was also expressed in lt-hESNSCs, although at low levels, which is consistent with the self-renewing status of these stem cells. Expression patterns of many Notch pathway components in lt-hESNSCs were comparable with those in fetal human brain. However, whereas transcripts for all four Notch receptors were detected in human brain, lt-hESNSCs expressed only NOTCH1, NOTCH2, and NOTCH3—a feature also shared by their parental hESCs (supporting information Fig. 1B). In contrast, the pluripotency marker Octamer-binding transcription factor OCT4 and the proneural gene ASCL1 were differentially expressed in hESCs and lthESNSCs. Furthermore, the human homologue of Hey2 was absent in lt-hESNSCs but expressed in H9.2 and I3 hESCs (supporting information Fig. 1B). In general, the high expression of Notch targets suggests that this pathway is active in lt-hESNSCs under self-renewing conditions. This notion is further supported by Western blot detection of the cleaved intracellular domain of NOTCH1 (NICD) with a cleavage site-specific antibody (Val1744, Fig. 1A, NSCs). Notch Activity Is Required for Maintenance of Self-Renewing Lt-hESNSCs and Efficiently Inhibited by DAPT Notch signaling can be blocked interfering pharmacologically with c-secretase activity. In this study, we used the GSI DAPT, which was previously shown to phenocopy Notch mutations [11, 12]. On DAPT treatment, release of NICD was abolished in lt-hESNSCs (Fig. 1A). Consistent with the inhibition of NICD formation, transcriptional levels of the Notch targets HES5 and HEY1 were significantly decreased (Fig. 1B, 1C). The radial glial marker and Notch target brain lipid-binding protein (BLBP) [13], which is highly expressed in lthESNSCs (supporting information Fig. 1A), was also significantly decreased on DAPT treatment. Conversely, expression of the proneural genes ASCL1 and Neurogenin-2 (NEUROG2), both targets of Hes/Hey-mediated transcriptional repression, was increased (Fig. 1B, 1C). Promoter analyses revealed that Hes3, in contrast to Hes1 and Hes5, is not direct target of Notch [14]. Accordingly, no significant differences in HES3 expression were detected on DAPT treatment (Fig. 1B, 1C). Interestingly, the regulation of HES1 in lt-hESNSCs appeared only minimally affected by Notch (Fig. 1B, 1C). This observation is in agreement with other data showing that Hes1 expression does not exclusively depend on Notch signaling [15] but, depending on the cell context, may also be regulated by other pathways [16, 17]. The results of several studies implicate Notch as key regulator of NSC maintenance and self-renewal, whereas its inhibition is associated with premature neuronal differentiation [5, 6]. We aimed at assessing the effect of DAPT-mediated Notch Inhibition in Human Neural Stem Cells Notch inhibition on the stem cell properties of lt-hESNSCs. Under self-renewing conditions (i.e., in the presence of FGF2 and EGF), 1.6% 6 0.1% of lt-hESNSCs showed spontaneous differentiation into b-III tubulin-positive neurons (Fig. 1D, untreated). DAPT induced a significant increase to 8.9% 6 1% b-III tubulin-positive cells already after 4 days (Fig. 1F, 1G). Glial differentiation markers were not detected at this early time point in any of the conditions tested (data not shown). Concomitantly with an induction of neuronal differentiation, a significant reduction in BrdU incorporation was observed in DAPT-treated lt-hESNSCs, compared with untreated or DMSO-treated cultures (Fig. 1D0 –1F0 , 1H). As Notch is not the only substrate of c-secretase activity, we assessed whether the DAPT-induced phenotype in lthESNSCs was indeed due to inhibition of Notch signaling. To that end, we performed a rescue experiment using a cell permeable protein containing Notch1-ICD linked to cell-penetrating peptide derived from trans-activator of transcription (TAT) from the human immunodeficiency virus (NICD-TAT; Haupt et al., submitted). When applied to lt-hESNSCs together with DAPT, NICD-TAT largely abolished the precocious neuronal differentiation induced by the GSI (supporting information Fig. 2A, 2C0 -2D0 ) and maintained BrdU incorporation at levels similar to untreated controls (supporting information Fig. 2B). These data show that the effects of DAPT on proliferation and differentiation of lt-hESNSCs are due to impaired Notch signaling and that inhibition of Notch is sufficient to shift lt-hESNSCs from self-renewal to commitment to neurogenesis. Notch Inhibition Affects Lt-hESNS Cell Cycle Progression To assess whether DAPT-mediated Notch inhibition is associated with changes in cell cycle progression of lt-hESNSCs, we employed flow cytometry and monitored their allocation to different cell cycle phases under self-renewing conditions (þFGF2/EGF) in the presence or absence of DAPT at 24, 48, and 72 hours. Notch inhibition increased the percentage of cells in G0/G1 at the expense of S-phase, with a difference that was significant at each time point (Fig. 2A). Differences in G2/M phase distribution between DAPT-treated and untreated lt-hESNSCs were minimal. Compared with control cultures, DAPT-treated lt-hESNSCs showed no reduction in the number of cells positive for Ki67—a marker of proliferation present during G1, S, G2, and M phases but absent from resting cells (G0) (supporting information Fig. 3A–3E). Taken together, these data indicate that DAPT-treated lt-hESNSCs slow down their progression through G1. Notch was shown to play a direct role in the regulation of G1/S-phase transition in the cell cycle of 3T3 fibroblasts, mouse hematopoietic cells and T-acute lymphoblastic leukemia (T-ALL) cells via the transcriptional induction of the S-phase kinase-associated protein two (SKP2) gene, which encodes an E3 ubiquitin ligase that targets p27Kip1 for degradation, thus allowing entry into S-phase [18, 19]. We therefore tested whether the expression levels of known regulators of G1 phase, as well as of the other cell cycle phases, are affected by DAPT. Western blotting analyses of lt-hESNSCs cultured under self-renewing conditions (þFGF2/EGF) revealed an upregulation of the cell cycle inhibitor p27Kip1 after 48 and 96 hours of DAPT treatment (Fig. 2B). Moreover, both qRT-PCR and Western blotting analyses showed that DAPT also induces a significant reduction of SKP2 levels (Fig. 2C, 2D). These data suggest that the impact of Notch on G1/S transition reported in other systems [18, 19] might also apply to our lt-hESNSCs. Borghese, Dolezalova, Opitz et al. 959 Figure 2. DAPT treatment impacts on the cell cycle of long-term self-renewing human embryonic stem cell–derived neural stem cells (lt-hESNSCs). (A): Histograms showing the relative distribution of lt-hESNSCs across the different cell cycle phases under self-renewing conditions, in the presence (þ) or absence (–) of DAPT at different days in culture. *, p < .05. (B): Western blot analysis of different cell cycle regulators in lt-hESNSC protein extracts after 48 and 96 hours of culture in the presence (þ) or absence (–) of DAPT. Alpha-tubulin was used as protein loading control. Images are representative of three independent analyses. (C): Quantitative real-time reverse transcription-polymerase chain reaction analysis of SKP2 transcript levels in lt-hESNSCs treated for 48 hours with DAPT (þ) compared with untreated control cultures (–) (equal to 1). Data are normalized to 18S rRNA levels and presented as mean þ SEM (n ¼ 3). **, p < .05. (D): Western blot analysis of SKP2 protein levels in lt-hESNSCs after 48 hours of culture in the presence (þ) or absence (–) of DAPT. Abbreviation: DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. We further detected an upregulation in the protein levels of both cyclin D1 and cyclin D2 after DAPT treatment (Fig. 2B), which is consistent with the observed accumulation of lthESNSCs in G1. Progression through G1 and transition to Sphase depend on the formation of complexes between D-type cyclins and their catalytic partners, the cyclin-dependent kinases CDK4 and CDK6. In contrast to the upregulation of cyclin D1 and D2, we detected a severe decrease in the protein levels of CDK4 and a decrease in the levels of CDK6 on DAPT treatment, both after 48 and 96 hours (Fig. 2B). This is in agreement with recent work showing that in T cells and in T-ALL cells Notch contributes to G1/S-phase progression by promoting the expression of CDK4 and CDK6 [20]. Although qRT-PCR analyses in lt-hESNSCs showed a downregulation of cyclin D3 transcript levels 48 and 96 hours after DAPT treatment (data not shown), these changes were not reflected at the protein level (Fig. 2B). Interestingly, DAPT also induced an upregulation of cyclin E1 protein (Fig. 2B). This may be in line with the observed accumulation of DAPT-treated lt-hESNSCs in G1, as cyclin E1 is known to play a role in late G1 phase. However, it might as well be that the upregulation of cyclin E1 is more directly associated with neuronal fate determination, as suggested from other studies [21]. We did not see differences in cyclin A2 protein levels (Fig. 2B), which is in accordance with the observation that no changes in G2/M were detected in DAPT-treated lthESNSCs. DAPT also led to a strong reduction in cyclin B1 protein levels (Fig. 2B). As no apparent changes in G2/M were induced, this effect might be explained by a change in the proportion of time the DAPT-treated cells spent in transition to metaphase and in metaphase itself. www.StemCells.com DAPT-Induced Notch Inhibition, in the Absence of FGF2/EGF, Accelerates Differentiation of Lt-hESNSCs into Functional Neurons Lt-hESNSCs can be propagated in culture in the presence of FGF2/EGF [3]. After 4 days of FGF2/EGF withdrawal, the number of cells positive for beta III-tubulin was similar to that observed on DAPT treatment in the presence of growth factors (Fig. 3A, 3C). However, when withdrawal from FGF2/ EGF was combined with DAPT, the number of b-III tubulinpositive cells increased to 62% 6 7.5%, which significantly exceeds the sum of the individual effects induced by DAPT or FGF2/EGF withdrawal alone (Fig. 3B–3C). This suggests that in lt-hESNSCs Notch and FGF2/EGF might act synergistically to prevent neuronal differentiation. Differentiation of lt-hESNSCs is performed by culture on matrigel-coated dishes in differentiation medium devoid of FGF2/EGF, as previously described [3]. After 2 weeks, cultures subjected to both FGF2/EGF withdrawal and DAPT treatment, but not cells subjected to FGF2/EGF withdrawal alone, had already formed prominent neuronal clusters connected by fasciculated neurites (Fig. 4A, 4C). Similar architectures were observed in cells subjected to FGF2/EGF withdrawal for 4 weeks (Fig. 4B). Simultaneous FGF2/EGF withdrawal and DAPT treatment consistently yielded more mature neuronal cultures with prominent fascicles of neurites (Fig. 4D). To assess functional maturation of DAPT-treated cells, whole cell currentclamp measurements were performed in 2-week-old cultures. Two-week-old DAPT-induced neurons proved to be still rather immature and electrically compact according to high input resistance (Rm) and low membrane capacitance (Cm) (Rm ¼ 2.06 6 0.46 GX; Cm ¼ 27.1 6 3.2 pF; resting membrane potential, 960 Figure 3. Combination of DAPT treatment and growth factor withdrawal largely enhances neuronal differentiation of long-term self-renewing human embryonic stem cell–derived neural stem cells (lt-hESNSCs). (A, B): Immunofluorescent detection of b-III tubulin in lt-hESNSCs after 4 days of FGF2/EGF withdrawal only (A) and FGF2/EGF withdrawal in combination with DAPT treatment (B). DAPI was used as nuclear counterstain (A0 , B0 ). Scale bars ¼ 100 lm. (C): Quantification of b-III tubulin-positive cells after 4 days in the presence or absence of FGF2/EGF and DAPT. Data are presented as mean þ SEM (n 4). *, p < .01. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; EGF, epidermal growth factor; FGF2, fibroblast growth factor-2. RMP ¼ –63.3 6 1.28 mV; n ¼ 10), comparable with early neuronal ontogeny in cerebral cortex [22]. However, voltage-clamp analysis revealed the presence of both fast transient Naþ and delayed rectifier-type Kþ currents (Fig. 4E). Furthermore, 2week-old DAPT-induced neurons were able to fire repetitive action potentials (Fig. 4E0 ), whereas control cells gained this ability only after 4 weeks of cultivation. The current profiles of DAPT-induced neuronal cells became more typical of mature neurons after 4 weeks, and most cells acquired an additional Atype Kþ current (Fig. 4F). In contrast, currents recorded from neurons derived from untreated cells reached the level of maturity seen in 2-week-old DAPT-treated cultures only after 4 weeks (Fig. 4G). Transient In Vitro Pretreatment with DAPT Promotes Differentiation of Grafted Lt-hESNSCs Considering the impact of Notch inhibition on the in vitro differentiation of our lt-hESNSCs, we wondered whether a transient treatment with DAPT could impact on the differentiation of transplanted NSCs. Such an acceleration of neuronal differ- Notch Inhibition in Human Neural Stem Cells entiation might be of significant translational value, since transplantation of human NSCs is complicated by the prolonged time required for their maturation [23–25]. We first assessed the differentiation of DAPT-treated cells on organotypic hippocampal slice cultures from neonatal rats. This experimental system has been successfully used to assess integration and differentiation into host tissue under controlled in vitro conditions [9]. For this experiment, we used lt-hESNSCs stably expressing GFP. On the basis of the observation that DAPT treatment in the absence of FGF2/EGF resulted in a strong increase of neuronal differentiation already within 3–4 days, we predifferentiated GFP-expressing lt-hESNSCs in vitro for 3 days in the presence or absence of DAPT. We then deposited them on 1-week-old hippocampal slice cultures, which were further maintained for 4 weeks. After this time, both control and DAPT-pretreated cells showed morphological evidence of neuronal differentiation with extensive axonal outgrowth across large areas of the slices (Fig. 5A– 5D). To assess the extent of neuronal maturation, we used a human-specific antibody to synaptophysin, a presynaptic marker associated with later stages of neuronal differentiation. In contrast to untreated cells (Fig. 5C0 ), lt-hESNSCs pretreated with DAPT were strongly positive for human synaptophysin (Fig. 5D0 ). Thus, transient in vitro treatment with DAPT before deposition onto hippocampal slices is sufficient to commit lt-hESNSCs to neuronal differentiation and to promote faster synaptic maturation of lt-hESNSCs. Finally, we evaluated whether the accelerated differentiation of DAPT-treated lt-hESNSCs could also be translated to an in vivo setting. We transplanted GFP-expressing lthESNSCs, predifferentiated for 3 days in vitro in the presence or absence of DAPT, into the mid-striatum of 9- to 12-weekold SCID-beige mice. Four weeks after transplantation, both DAPT-pretreated and control cells exhibited strong and indistinguishable human doublecortin staining across the entire graft, as quantified in Figure 5G. However, 2 weeks after grafting, only cells pretreated with DAPT revealed strong human doublecortin staining (Fig. 5F), whereas grafts containing untreated cells (Fig. 5E) barely exhibited any specific signal above background levels (Fig. 5E–5G). This result is compatible with the notion that DAPT treatment accelerates the onset of neuronal differentiation without interfering with overall neuronal lineage commitment. DISCUSSION In this work, we assessed the role of Notch signaling in a population of long-term expandable hESNSCs. We characterized the expression of components of the Notch pathway and showed that ligands, receptors, as well as targets of Notch are represented in lt-hESNSCs. We further demonstrated that Notch is active in lt-hESNSCs, and its activity is required to maintain these cells in an undifferentiated/self-renewing state. Conversely, interfering with Notch signaling in lt-hESNSCs by means of the GSI DAPT affected expression of several cell cycle regulators, slowed down G1/S-phase transition and promoted early onset of neurogenesis. We exploited the possibility of modulating Notch signaling to gain control on the differentiation of lt-hESNSCs and devised a protocol to accelerate the in vitro derivation of functional neurons. We further assessed the impact of transient treatment with DAPT on the differentiation of transplanted lt-hESNSCs and showed that short-term exposure to a GSI in vitro is sufficient to accelerate the onset of neuronal differentiation of engrafted cells in vivo. Borghese, Dolezalova, Opitz et al. 961 Figure 4. Functional maturation of neurons differentiating from DAPT-treated long-term self-renewing human embryonic stem cell–derived neural stem cells (lt-hESNSC) cultures. (A–D): Immunostaining with an anti-b-III tubulin antibody of neuronal cultures from lt-hESNSCs after 2 and 4 weeks of in vitro differentiation (FGF2/EGF), in the presence and absence of DAPT. Scale bars ¼ 100 lm. (E, E0 ): Representative whole cell current profiles and action potentials recorded from DAPT-induced neurons after 2 weeks of in vitro differentiation (n ¼ 5). (F, G): Representative whole cell current profiles of neurons derived from lt-hESNSC cultures after 4 weeks of in vitro differentiation in the presence (F) and absence (G) of DAPT. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; EGF, epidermal growth factor; FGF2, fibroblast growth factor-2. Much insight in the role of Notch during human early neural development has been gained in recent years. This signaling pathway was shown to contribute to the commitment of hESCs toward the neural lineage [26], and to the maintenance of primitive NSCs derived from these human pluripotent cells [25]. In particular, Elkabetz et al. showed that maintenance of rosette-forming NSCs isolated at early stages of hESC differentiation strongly depends on Notch. However, these cells appeared difficult to proliferate in culture; their expansion in the presence of FGF2/EGF eventually led to an adherently growing type of NSCs with restricted differentiation potential [25]. These observations implied that long-term expansion of human NSCs is associated with a loss of in vitro patternability. The lt-hESNSCs recently identified in our laboratory and used in this study represent an intermediate between these two NSC stages. Although they can be continuously maintained in culture in presence of FGF2/EGF, they retain the ability to form rosette structures typical of early neuroepithelial cells and remain amenable to morphogens mediating, for example, generation of mesencenphalic dopamine neurons and spinal cord motoneurons [3]. The fact that maintenance of both the rosette cells described by Elkabetz et al. and our lt-hESNSCs depends on Notch signaling further supports a close relationship of the two cell populations. The findings accumulated by these and additional studies [27–31], together with the recent finding that neuroepithelial rosette cultures recapitulate several aspects of embryonic neural tube development [32], demonstrate a fundamental requirement of Notch at different stages of NSC development, with a function in NSC maintenance that appears evolutionary conserved across vertebrate species, including humans. Notch activity. We found that DAPT-treated cells accumulate in G1 phase. In the mouse ventricular zone, progression from proliferative to neurogenic divisions was found to be associated with changes in the length of the cell cycle, especially of G1 [34]—a phase where cells are considered more sensitive to differentiation signals [35]. Furthermore, there is evidence that lengthening of G1 triggers neurogenesis [36–38]. In particular, it has been shown that overexpression of CDK4/ CyclinD1 shortens G1 and inhibits neurogenesis, thereby promoting expansion of mouse neural progenitors in vivo, whereas CDK4/CyclinD1 downregulation triggers G1-lengthening and induces neurogenesis [38]. We found that treatment of lt-hESNSCs with DAPT induced a strong decrease in the protein levels of CDK4 and, to a lower extent, CDK6, alongside with the accumulation of cells in G1. This is in line with the finding that Notch contributes to G1/S-phase transition of mouse T cells and human T-ALL cells via the regulation of CDK4 and CDK6 protein expression [20]. In agreement with recently published data [18, 19], we further show that p27Kip1 protein levels were strongly increased on Notch inhibition, whereas the expression of SKP2, coding for the F-box subunit of the ubiquitin ligase complex that targets this cell cycle inhibitor for degradation, was decreased. However, it remains to be determined whether in lt-hESNSCs the DAPT-mediated upregulation of p27Kip1 contributes to the shift from proliferation to differentiation via a mere inhibitory effect on cell cycle progression or through a more direct role in the induction of neurogenesis, as suggested by data from other vertebrate systems [39, 40]. In light of what is known so far, our data support the hypothesis that Notch plays a key role in the maintenance of human NSCs and that this effect is, at least in part, based on the control of the timing of G1/S-phase progression via the regulation of multiple G1-associated proteins. Notch-Dependent Regulation of Neural Stem Cell Cycle Notch Inhibition and FGF2/EGF Withdrawal Synergize in Promoting Neuronal Differentiation The cell cycle has been proposed to serve as ‘‘gatekeeper’’ to self-renewal [33], and Notch has been implicated in cell cycle control. We assessed how Notch signaling might regulate the rate at which lt-hESNSCs commit to neurogenesis by investigating cell cycle changes occurring on interference with Withdrawal of FGF2 and EGF from the culture medium is well known to trigger differentiation of lt-hESNSCs and other NSCs. Here, we show that Notch inhibition in the presence of FGF2/EGF, too, suffices to promote differentiation. Interestingly, when both conditions are combined, a very large Conserved Role of Notch in Human NSCs www.StemCells.com 962 Notch Inhibition in Human Neural Stem Cells Figure 5. Transient exposure to DAPT in vitro accelerates differentiation of long-term self-renewing human embryonic stem cell–derived neural stem cells (lt-hESNSCs) grafted onto hippocampal slice cultures (A–D) or into the adult mouse striatum (E, F). (A, B): Immunofluorescence images showing differentiating anti-GFP-labeled lt-hESNSCs untreated (control, A) or transiently in vitro pretreated with DAPT (B) 4 weeks after deposition on rat hippocampal slice cultures. Insets in (A) and (B) correspond to areas where images (C) and (D), respectively, were taken. (C, D): Assessment of human synaptophysin immunostaining intensity in untreated (C, C0 ) and DAPT-pretreated (D, D0 ) lt-hESNSC-derived neurons. (E, F) Immunofluorescence images showing 2-week-old striatal grafts derived from lt-hESNSCs predifferentiated in vitro in the absence (E, E0 ) or presence (F, F0 ) of DAPT, stained for human doublecortin and GFP. Dorsal is top and ventral is bottom. (G): Quantitative assessment of doublecortin immunostaining intensity in 2- and 4-week-old grafts from lt-hESNSCs predifferentiated in vitro in the presence (þDAPT) or absence of DAPT (*, p < .05). Quantitative data are presented as mean þ SEM (for quantitative analysis, see also ‘‘Materials and Methods’’ section). Scale bars: (A, B) ¼ 1 mm, (C0 , D0 ) ¼ 100 lm, (E0 , F0 ) ¼ 200 lm. Abbreviations: GFP, green fluorescent protein; DAPI, 40 ,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. number of lt-hESNSCs commit to neurogenesis, at a rate that is significantly higher than the sum of the individual effects. This would suggest that Notch and FGF2/EGF-mediated signaling act in synergy to prevent premature differentiation of lt-hESNSCs. Such a conclusion is not unexpected considering that several lines of evidence from different systems have already pointed at interactions of Notch with other pathways— including FGF and EGF signaling—in the context of NSC biology [41–45]. Lt-hESNSCs represent at date a readily accessible system to model in vitro early human neural development and a versatile and stable cell source for biomedical and pharmaceutical applications. A clear limitation associated with lthESNSCs and human NSCs in general is the long time required for the differentiation of defined cell types, which is in line with the longer time span of human development compared with other vertebrate systems. The combination of DAPT-mediated Notch inhibition and growth factor Borghese, Dolezalova, Opitz et al. withdrawal significantly shortened the time needed for differentiation of lt-hESNSCs into functional neurons. Electrophysiological recordings showed that already after 2 weeks of in vitro differentiation lt-hESNSC-derived neurons were able to fire action potentials—a degree of functional maturation that is, in this cell population, observed only after several weeks of in vitro differentiation in untreated cultures [3]. Modulating Notch signaling with DAPT or similar GSIs might thus facilitate the generation of hESC-derived functional neurons for pharmaceutical screening and other biomedical applications. Interestingly, a transient inhibition of Notch proved to be sufficient to commit lt-hESNSCs to differentiation. This observation is in agreement with a recently published study showing that short-term treatment with DAPT leads to a permanent commitment of chicken retinal progenitor cells to differentiation [46]. We showed that lt-hESNSCs pretreated with DAPT for 3 days and subsequently ‘‘transplanted’’ onto hippocampal slice cultures yielded neurons expressing much higher levels of the presynaptic protein synaptophysin than untreated control cells. We further showed that transient DAPT-treatment of lt-hESNSCs in vitro significantly accelerated the onset of neuronal differentiation upon grafting into the mouse striatum. Promoting neuronal differentiation of grafted NSCs by transient pregrafting exposure to GSIs such as DAPT might have significant impact on the development of cell replacement therapies. The protracted differentiation times observed in human neural cells can lead to excessive proliferation after transplantation, resulting in a phenomenon frequently referred to as ‘‘neural overgrowth’’ [23–25]. GSI treatment could permit better control on donor cell differentiation, thereby providing a way to avoid overgrowth in the host. Since systemic administration of GSIs is known to elicit adverse side effects [47], the efficacy of transient in vitro pretreatment documented here might eventually point to a route, which bypasses these side effects while promoting neural transplant maturation. On the other hand, a mere pretreatment with GSIs would not be expected to influence the very protracted functional integration of grafted NSCs, which can take up to several months [3]. However, systemic application of GSIs might, despite its side effects, represent an avenue worthwhile pursuing to explore acceleration of functional maturation of grafted NSCs in vivo. REFERENCES 963 CONCLUSION Our data prove that Notch activity is required for maintenance of long-term self-renewing human ESC-derived neural stem cells, whereas inactivation of Notch signaling promotes early onset of neuronal differentiation. This effect might be in part explained with a role of Notch in the control of the timing of G1/S phase transition. We made use of the effect induced by disruption of Notch signaling and exploited it to expedite neuronal maturation of hESNSCs both in vitro and in vivo. Recently, it has been shown by Chambers et al. that dual inhibition of SMAD signaling permits direct and potent generation of neural precursors from human pluripotent stem cells, thereby allowing for the subsequent derivation of neurons within a short time period (48). It would be interesting to assess whether combined inhibition of SMAD and Notch signaling could provide a way to further accelerate the generation of functional neurons from human pluripotent cells. ACKNOWLEDGMENTS We thank Prof. Joseph Itskovitz-Eldor (Technion, Israel Institute of Technology, Haifa, Israel) for providing the hESC lines I3 and H9.2, Dr. Elmar Endl, Institute of Molecular Medicine, University of Bonn for FACS analyses, Dr. Didier Trono (EPFL, École Polytechnique Fédérale de Lausanne, Switzerland) for providing the pLVTHM vector, Katja Hamann, Christina Leufgen, and Monika Endl for outstanding technical support and Dr. Winfried Barchet, Dr. Sandra Blaess, Dr. Julius Steinbeck, Laura Mürtz, and Beate Syttkus for critical reading of the manuscript. This work was supported by the EU (LSHG-CT-2006-018739, ESTOOLS; FP7-HEALTH-2007-B-22943-NeuroStemcell), the DFG (SFB TR3 D2), MSM0021622430, AV0Z50390703, 1M0538, and the Hertie Foundation. 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