Download Inhibition of Notch Signaling in Human Embryonic Stem CellDerived

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).
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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.
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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.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
O.B. has stock in and is a cofounder of LIFE & BRAIN GmbH.
The other authors have no financial interests to disclose.
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