Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells
- Sun-Mi Woo†1,
- Janghwan Kim†1,
- Hyo-Won Han1,
- Jung-Il Chae1, 2,
- Mi-Young Son1,
- Sunwha Cho1,
- Hyung-Min Chung2,
- Yong-Mahn Han3 and
- Yong-Kook Kang1Email author
© Woo et al; licensee BioMed Central Ltd. 2009
Received: 5 November 2008
Accepted: 17 August 2009
Published: 17 August 2009
Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.
From hESCs, we derived NESs, the in-vitro version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of in-vivo-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their in-vivo counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a γ-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons.
Our results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an in-vitro model for in-vivo neurogenesis.
Neural stem cells (NSCs), which have properties of self-renewal and differentiation into neurons and glias, are usually isolated from fetal and adult brains in the form of floating clonal derivatives of the NSCs placed in culture, known as neurospheres (NSs) [1–3]. NSCs have the potential to be used in cell replacement therapy for neural disorders such as Parkinson's disease and Alzheimer's disease as well as other neurological disorders including spinal cord injuries . For therapy, maintenance and expansion of the NSCs are necessary to provide sufficient amount of cells for patients to be treated.
Human NSCs can be obtained from brains and from human embryonic stem cells (hESCs) by a step-wise differentiation procedure [5–8], and such hESC-derived NSCs are usually cultured as NS-like aggregates. However, the NS-like aggregates, also called neuroectodermal spheres (NESs; ), differ from the NSs in that NESs have a distinctive radial cluster of columnar epithelial cells, called a rosette [8, 10]. The rosette resembles a developing neural tube and contains multipotential neuroprogenitor cells that have a similar expression profile as the neuroepithelial cells in the neural tube. Analyses of microarray data revealed that brain-derived NSCs and hESC-derived NSCs were shown to express distinct groups of genes and, nonetheless, they did share many properties involving NSC markers [11, 12]. In addition, the brain-derived NSs generally exhibit specific regional markers along with dorso-ventral and antero-posterior axes and, of them, hESC-derived NESs tend to preferentially express markers of anterior neural ectoderm . Together, NESs might be assumed to mimic the pattern of in vivo neurogenesis to a degree .
It is well known that Notch signaling has a role in deciding cell fates during development . With regard to neural development, Notch signaling also has an important role in the maintenance of neural stem-cell features. Notch1, Presenilins and RBP are key Notch signaling molecules – a receptor, a regulator and a co-effector, respectively. In the fetal brains of Notch1-/-, Presenilins-/-, or RBP-Jκ-/- mice, NSC levels were shown to be completely depleted . Also, inactivation of Notch-regulated genes such as Hes1 and Hes5 induced a premature neuronal differentiation during brain development . Studies with Presenilin-deficient mice have shown that Notch signaling is necessary to maintain all NSCs, regardless of their locations in the brain or age of the mouse . Several studies have provided important findings about the roles of Notch signaling in neural development; unfortunately, however, most of these studies have investigated the NSCs of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. Therefore, information on human NSCs is scarce, which prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. hESC-derived NSCs could possibly replace the rare human NSCs , which highlights the importance of the study to characterize the complicated, web-like molecular events, including Notch signaling, that occur in the in vitro-produced NESs.
In this study, we investigated the role of Notch signaling in hESC-derived NESs. We first verified that hESC-derived NESs had features similar to neurospheres derived in vivo. We demonstrated that Notch-related molecules were expressed at higher levels in the NESs than in the hESC-derived embryoid bodies. Furthermore, when Notch signaling was inhibited by a specific inhibitor for the γ-secretase, the rosette folds were not visible, and the self-renewing activity and the proliferative potential were significantly reduced in the resulting NESs. These observations indicate that Notch signaling is active in the NESs, and, to our knowledge, this, along with a recent paper by Elkabetz et al. , is the first description about the role of Notch signaling in maintaining self-renewal of NSCs derived from hESCs.
Human embryonic stem cell (hESC) culture
CHA-hES3  was maintained on mitomicin C-treated STO feeder cells (ATCC, Manassas, Virginia, USA). H9  (WiCell, USA) was maintained on γ-irradiated mouse embryonic fibroblasts in gelatin-coated culture dishes at 37°C, 5% CO2 in air. These hESCs were sub-cultured by mechanical section using a hand-made glass pipette. Human ESCs were cultured in DMEM/F12 containing 20% serum replacement, 0.1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 U/ml penicillin-streptomycin and 4 ng/ml basic fibroblast growth factor (bFGF)(All from Invitrogen, Carlsbad, USA). Culture media were replenished everyday. Our research was performed under ethical approval from the Institutional Review Board (IRB) at KRIBB.
Generation and culture of Neuroectodermal spheres (NESs) from hESCs
Human ESC colonies were dissected into 500 μm squares by tissue chipper  or ESCD with 500 μm pattern , transferred to plastic Petri dishes containing EB medium (hESC medium plus 10% serum replacement without bFGF), and cultured for 7 days. EB medium was then replaced to neuroectodermal sphere medium (NSM; DMEM:F12(1:1 in volume), B27 supplement (1:50), N2 supplement (1:100), 100 U/ml penicillin-streptomycin, 20 ng/ml bFGF, 20 ng/ml human epidermal growth factor (EGF) (all from Invitrogen) and 10 ng/ml human leukemia inhibitory factor (LIF, Sigma, St. Louis, MO)). A half medium was refreshed in every 48 hours. NESs were sub-cultured using McIlwain tissue chopper (Mickle Engineering, Gomshall, Surrey, UK) when they were grown to 500 μm in diameter . Volume of NESs was measured by the formula for the volume of sphere, (4/3)πr3; radiuses of individual spheres were determined by taking mean length of long and short axis.
Total RNA was isolated from hESCs, EB and NESs using RNesay kit (Qiagen, Valencia, UAS) and reverse-transcribed into cDNA with Superscript First Strand Synthesis System (Invitrogen) using oligo-d(T) primer as described in the manufacture's instructions. As a reference, the transcripts of GAPDH (for glyceraldehydes-3-phosphate dehydrogenase) or β-actin gene were amplified. Sequence information of primers and the lengths of amplified products are seen in Additional file 1. Primers for amplifying CNS marker genes are listed elsewhere . Amplification conditions were as follows: single cycle of 94°C for 5 min followed by 30 – 35 cycles of 94°C for 30 s, 56–60°C for 30 s and 72°C for 30 s, and the final single cycle of 72°C extension for 7 min. Products were analyzed on 1.5% agarose gel and visualized by ethidium bromide staining.
Floating NESs were plated on the matrigel (BD Biosciences, San Jose, CA)-coated dishes. The NESs attached were fixed with 10% formalin solution (containing 4% formaldehyde, Sigma) for 20 min followed by permeabilization for 30 min in PBS containing 0.1% Triton X-100. After blocking with 4% normal donkey serum (Molecular Probes, Eugene, OR, USA) for 1 hour, the samples were incubated with following primary antibodies at 4°C overnight: antibodies for Nestin (1:200, Chemicon, Temecula, CA), PAX6 (1:50, DSHB, Iowa City, Iowa, USA), NOTCH1 (bTAN20, 1:50, DSHB), DLL1 (1:200, Santa Cruz Biotechnology, CA, USA), TUJ1 (1:500, Covance, Madison, Wisconsin, UAS), JAG1 (1:50, Santa Cruz Biotechnology), N-Cadherin (1:50, Santa Cruz Biotechnology). Primary antibodies were detected by using Cy2- or Cy3-conjugated Donkey anti-Goat, anti-rabbit or anti-mouse secondary antibodies (Jackson laboratories, West Grove, PA, USA) for 45 min at room temperature. After reaction with secondary antibodies, the cells were stained with 100 nM DAPI (4',6-diamidino-2-phenylindole)(Molecular Probes) for 5 min, and mounted. Fluorescence-labeled NESs were viewed under an IX51 Olympus fluorescence microscope (Olympus, Japan) or Axiovert 200M equipped with ApoTom (Carl Zeiss, Gottingen, Germany).
Neuroectodermal sphere re-forming Assay
NESs were dissociated with 2 mg/ml collagenase into single cells and cultured in NSM containing 0.1% DMSO or 5 μM DAPT for 17~18 days at a density of 1 × 105 cells/ml. Fifty percent of medium was replaced every 4~5 days. NESs with sizes more than 50 μm were counted.
BrdU incorporation assay
Cells cultured in the NSM were treated with 10 μM 5-bromo-2'deoxyurine (BrdU; Roche Molecular Biochemicals, Sussex, UK) for 24 hours. Spheres were dissociated with collagenase and plated on the matrigel-coated coverslip for counting. Cells were fixed with formalin solution 10% (Sigma) for 15 min followed by permeabilization for 30 min in PBS containing 0.1% Triton X-100. DNA denaturation were performed by 2N HCl for 10 min and neutralized with 0.1 M Sodium tetra-borate for 10 min. Following procedures were the same as immunocytochemical method above mentioned. Genome-integrated BrdUs were detected using anti-BrdU antibody (1:200, BD pharmingen, San Diego, USA) and Cy3 conjugated anti-mouse secondary antibody. The proportion of BrdU positive cells relative to total cells counted was estimated under a fluorescent microscope.
Trypan blue staining
NESs cultured in the NSM containing 0.1% DMSO or 5 μM DAPT for 4 days were dissociated with 2 mg/ml collagenase into single cells. An equal volume of Trypan blue stain solution (0.4%, Invitrogen) was added to the cell suspension. After 5 min, trypan blue stained cells and total cells were counted using a hemacytometer under the IX51 Olympus inverted microscope (Olympus).
Quantification of TUJ1-positive cells in NESs
After 4 day culture in the NSM containing 0.1% DMSO or 5 μM DAPT, NESs were dissociated into single cells with 2 mg/ml collagenase and allowed to attach on the matrigel-coated coverslip. After immunostaining either with Nestin or TUJ1 antibody, the proportion of Nestin- or TUJ1-positive cells relative to the whole cells counted was calculated.
Western blot analysis
Antibodies against Jagged1 (JAG1, Santa Cruz Biotechnology), Delta-like-1 (Dll1; Santa Cruz Biotechnology), cleaved Notch1 (NICD; Cell Signaling Technology, Beverley, MA USA), Nestin (Chemicon), TUJ1 (Covance, Richmond, CA), MAP2 (Chemicon), S100 (Abcam, Cambridge, UK), GFAP (Novus Biologicals, Littleton, CO, USA), NG2 (Chemicon), CNPase (Chemicon), HES1 (Chemicon) and HES5 (Sigma) were used for Western-blot analyses. For protein extraction, cells were lysed in a buffer containing 20 mM HEPES, 50 mM NaCl, 10% glycerol, 0.5% Triton X-100 and 2% β-mercaptoethanol. Concentrations were determined by the Bradford method. The protein samples (20 μg) were separated by 6%, 8% and 15% SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell Inc., Keene, NH, USA) with Tris-glycine-methanol buffer (25 mM Tris, 192 mM glycine and 20% methanol). After blocking with the TBS buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 5% non-fat dry milk and 0.1% Tween20, the membrane was incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat antibody to rabbit IgG (Santa Cruz Biotechnology), and developed with enhanced chemiluminescence reagent (Amersham, Piscataway, NJ, USA).
Results and Discussion
Derivation and characterization of neuroectodermal spheres from human embryonic stem cells
The hESC-derived NESs attached to the Matrigel-coated culture dish were immunostained for neural stem cell (NSC) markers such as SOX1, PAX6 and Nestin. The rosettes of various sizes were positively stained for all these NSC markers (Figure 1C). In addition, the hESC-derived NESs were stained for a neuronal marker TUJ1, we found TUJ1-positive neurites sporadically scattered around the boundaries of NES clumps. Flow cytometry showed that more than 95% in both CHA-hES3- and H9-derived NESs were positively stained for the neural precursor cell surface marker PSA-NCAM (data not shown). When analyzed at the transcriptional level, the NESs showed increased expression levels of NSC marker genes such as NES, MSI1 and 2, PAX6, VIM, SOX1, and SOX3, whereas none of the mesoderm-lineage markers (Meso; VEGFR2, C-KIT, GATA2 and T) or the endoderm-lineage markers (Endo; AFP and GATA4) were transcribed in a NES-specific manner (Figure 1D). The transcripts for the ESC marker genes (Stem) OCT4 and NANOG, were undetectable in the NESs. The expression patterns of these NSC markers are similar to recent reports; for example, PAX6 expression continued in 7-day-old EBs, whereas SOX1 expression began only after NES formation . RT-PCR results showed that anterior CNS markers such as FoxG1 and Otx2 were more expressed in the NESs than mid-hindbrain markers such as Pax2 and En1 and markers of posterior CNS fate such as Krox20 and HoxB4  (Figure 1E). This result agreed with a recent report , suggesting that in the absence of extrinsic patterning cue, NESs acquire markers defining anterior CNS identity. Taken together, these morphological, immunocytochemical, and molecular-level results demonstrate that the hESC-derived NESs are suitable as an in vitro model of human in vivo-derived neuroprogenitors.
Components of Notch signaling were up-regulated in hESCs-derived NESs
Inhibition of Notch signaling leads to a loss of the stem cell characteristics from the neuroectodermal spheres
To assess the proliferating capacity of neuroprogenitors in the Notch signaling-inhibited NESs, we performed NES-reforming assay [15, 26, 39]. NESs were enzymatically dissociated into single cells and allowed to re-construct sphere colonies in NSM with or without DAPT. As shown in Figure 3E, the number of emerging spheres in the DAPT-treated cells was reduced to 25% that of the control group in CHA3 (of 105 cells plated, 29.7 ± 4.7 (mean ± standard error) and 7.9 ± 2.2 cells in DMSO control and DAPT-treated cells, respectively; p < 0.05) and H9 (55.1 ± 1.8 cells for control vs 13.0 ± 1.2 cells for DAPT-treated group; p < 0.005) cell line s. The reformation frequency of hESC-derived sphere colony has not previously been estimated with hESC-derived NESs, making it impossible to compare the reformation efficiency, but it seems to be about ten-fold less than the frequency (2.4%) of mouse neuroprogenitors derived in vivo . In addition, results of the bromo-2'-deoxyuridine (BrdU) incorporation assay showed that DAPT treatment reduced the proportion of replicating cells to 39% of the control group for CHA3 hESCs or 60% of the control group for H9 hESCs (Figure 3F). Together, these results show that the Notch signaling is mainly involved in the maintenance of rosette structures, the biochemical roles of which are probably linked to maintenance and self-renewal of the neuroprogenitor population in NESs.
Inhibition of Notch signaling drives neuroectodermal spheres to differentiate into neuronal cells
Moreover, withdrawal of growth factors (GFs) such as fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and epidermal growth factor (EGF)  from the NSM furthered differentiation of the hESC-derived NESs into neuronal cells (Figure 4D), agreeing with previous reports [42, 43]. In the absence of GFs, DAPT treatment resulted in accumulation of neurite bundles about NESs (see arrows in Day8 and Day12), in contrast to the samples treated with both GF and DAPT in which neurite bundles were hardly detected. These results coincided with the RT-PCR result in Figure 3D where Notch inhibition resulted in silencing HES1 and HES5 genes, which in turn continued to de-repress pro-neuronal genes such as NGN1 and MASH1 [29, 38]. Meanwhile, the GF-negative DMSO control at day 8 and 12 still possessed rosettes, the sizes of which, however, were tiny compared with those of GF-positive DMSO control. This leads to a speculation that small-sized rosettes are supposed to be gradually fused together to large-sized ones and, in this process, GFs play roles in facilitating the presumable fusion event among individual rosettes. Together, our results indicate that inhibition of Notch signaling disturbs the self-renewal of neuroprogenitors in the hESC-derived NESs and leads ultimately to differentiation to neuronal cells. However, we should keep in mind that the γ-secretase that is inactivated by DAPT not only cleaves Notch receptors but also other proteins (for review, see ). For confirmation that Notch inhibition indeed directs neuronal differentiation, evidence needs to be provided supporting that the DAPT effects on rosette structure can be rescued with exogenous NICD expression in DAPT-treated cultures, or be mimicked by knockdown experiments for NICD or RBP.
NSCs have considerable therapeutic values in cell-replacing regenerative treatment of currently incurable neural diseases. In addition, unlimited supply of functional human neurons is only possibly from NSCs, and this would allow a fast and effective high throughput screening for neural disease therapies. Human ESCs are undoubtedly the preferred source of NSCs. We have developed a simple method for deriving NSCs or neuroprogenitors from hESCs, with an emphasis on minimizing the degree of variation among individual EBs and sphere colonies through size regulation (Figure 1A). The use of sub-culture devices such as tissue chopper  or ESCD  allowed us to obtain EBs with regular sizes (Figure 1B) that form homogeneous NESs. Our method of NES derivation has the advantages of a short culture period, thus avoiding additional attachment and selection steps . This markedly simplifies the current NES derivation procedures [6, 8, 45, 46] without reducing the efficiency, which is necessary for the practical application of hESC-derived NSCs to cell therapeutics and drug screening. Our NES derivation protocol is similar to a recently reported protocol .
In vertebrate, activation of Notch signals inhibits neuronal differentiation and maintains the stem-cell characteristics of NSCs or neuroprogenitors derived in vivo . We investigated whether Notch signaling is active and therefore has a genuine role in the hESC-derived NESs, and we obtained several results. First, results of RT-PCR and Western-blot analyses showed that most of the known key components of the Notch signaling pathway such as receptors (NOTCH1, NOTCH2 and NOTCH3), ligands (DLL1, DLL3 and JAG1), and regulators (NICD, MIB1, MIB2 and PSEN1) were abundantly expressed in the NESs at the protein and mRNA levels (Figure 2A and 2B). Second, the expression levels of Notch signal members and the resulting target genes (HES1 and HES5) were increased in the NESs compared with those in the EBs. This was particularly true for the NICD, DLL1, JAG1 and HES1 (Figure 2B). Third, immunostaining of the NESs for the plasma-membrane-bound ligand JAG1 and DLL1 demonstrated that both are localized mainly to the cells comprising inner rims of the rosettes, rather than being expressed throughout the NESs (Figure 2C). Fourth, the treatment with Notch inhibitor DAPT removed rosette structures from both floating and attached NESs, and was associated with the reduction of NSC marker expressions and proliferation potential in the NESs (Figure 3). Finally, DAPT treatment induced neurite formation and increased expression of TUJ1 (Figure 4), indicating that Notch inhibition drives the NESs to differentiate preferentially into neuronal cells, in agreement with the observation that Notch-inhibited neuroprogenitor cells favor differentiation toward neuronal cells in vertebrate and invertebrate (for review, see ). Therefore, we concluded that Notch signaling actively functions in the NESs or, more specifically, in the rosettes, and that Notch signaling is responsible for maintenance of the stem-cell features of NSCs or neuroprogenitors in the rosettes. Therefore, our results indicate that the hESC-derived NESs or the neural rosettes are a good in-vitro model for neurogenesis in vivo.
NSCs have considerable therapeutic values in cell-replacing-regenerative treatment of currently incurable neural diseases. hESCs are one of the best sources of NSCs or neuroprogenitor cells owing to their unlimited proliferation. In this study, we derived NESs containing neuroprogenitors from hESCs, and verified that these hESC-derived NESs were typical of neurospheres burying neuroprogenitors and were characteristic of activated Notch signaling. DAPT-induced inhibition of Notch signaling led to loss of the stem-cell characteristics from the NESs and drove them to differentiate into neuronal cells. These results are the first to demonstrate the roles of Notch signaling in hESC-derived NESs with biochemical features similar to those in neurospheres derived from animal brains, or fetal or adult human brains. Therefore, the hESC-derived NESs or neural rosettes are considered to be a good in vitro model for studying the neurogenesis that occurs in vivo. We believe that our results might aid further study of the mechanisms by which rosettes form and expand in vitro, how neuroprogenitor cells maintain their stem-cell-like characteristics in the cell culture environment, and the stem-cell characteristics that lead to asymmetric division.
This research was supported by grants from KOSEF (2006-04082 and M10643020004-08N4302-00400).
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