STEM CELLS AND DEVELOPMENT Volume 21, Number 17, 2012 Mary Ann Liebert, Inc. DOI: 10.1089/scd.2011.0678
Glycogen Synthase Kinase-3 Inhibition Promotes Proliferation and Neuronal Differentiation of Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors Fereshteh Esfandiari,1,2 Ali Fathi,2,3 Hamid Gourabi,4 Sahar Kiani,1 Shiva Nemati,1 and Hossein Baharvand1,2
Human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) have the ability to self-renew and differentiate into glial and neuronal lineages, which makes them an invaluable source in cell replacement therapy for neurological diseases. Therefore, their enhanced proliferation and neuronal differentiation are pivotal features that can be used in repairing neurological injuries. One of the main regulators of neural development is Wnt signaling, which results in the inhibition of glycogen synthase kinase 3 (GSK-3). Here, we assess the impact of GSK-3 inhibition by the small molecule CHIR99021 on the expansion and differentiation of hiPSC-NPs in an adherent condition and a defined medium. Cell proliferation analyses have revealed that inhibition of GSK-3 in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) increased the proliferation of hiPSC-NPs across 10 passages. The inhibition of b-catenin signaling by XAV and NOTCH signaling by DAPT reversed CHIR impact on hiPSC-NPs proliferation. The target genes of b-catenin, C-MYC and CYCLIN D1 as well as NOTCH target genes, HES1 and HES5 were upregulated. The treatment of NPs by CHIR in the absence of bFGF and EGF resulted in an increase of neuronal differentiation rather than proliferation by stabilization of b-catenin regardless of the NOTCH pathway. Thus, GSK-3 inhibition has been shown to promote proliferation of the NPs by activating b-catenin and NOTCH-related cell cycle genes in the presence of bFGF and EGF. Additionally, during GSK-3 inhibition, an absence of these growth factors allows for the switch to neuronal differentiation with a bias toward a dopaminergic fate. This may provide desired cells that can be used in therapeutic applications and offer insights into the etiology of some neurological disorders.
Introduction
H
uman-induced pluripotent stem cells (hiPSCs) that have the ability for self-renewal and maintenance of pluripotency into differentiated cells of all 3 embryonic germ layers are derived from somatic cell reprogramming in vitro [1,2]. hiPSCs, which serve as a new, promising autologous source of neural progenitors (NPs) and neuronal cells have attracted much attention [3–6]. NPs have extensive capacity for proliferation, self-renewal, and differentiation into glial and neuronal lineages. They provide an invaluable potential to contribute to the regeneration of nervous system diseases (for review see Refs. [4,7]). Therefore, enhanced control of
hiPSC-NPs proliferation and differentiation into specific neural cell lineages plays a pivotal function in the repair of neurological injuries. In this regard, Wnt signaling is one of the main NP regulators [8] that predominantly leads to glycogen synthase kinase 3 (GSK-3) inhibition by the canonical pathway [9]. Wnt signaling mediates self-renewal in mouse NPs in vitro [10,11] and accelerates the proliferation of hippocampal NPs by shortening the cell cycle [12]. Activation of the canonical Wnt pathway causes an increase in expansion of NPs and suppression of neuronal differentiation, which has been shown in the early stages of chick spinal cord and mouse forebrain development, as well as in human embryonic stem
1 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 2 Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran. 3 Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 4 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
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3234 cells [13–16]. Conversely, Wnt3a can increase neurogenesis during human ReNcell cell line differentiation and is independent of the Wnt/b-catenin transcriptional activity [17,18]. Recently, accumulative evidence suggests that small molecules can replace growth factors [19–23]. Small molecules are low molecular weight chemically synthesized molecules that freely diffuse from the cell membrane. These molecules can reversibly alter the function(s) of a single protein (or multiple proteins) by exerting exquisite temporal control in the absence of genetic modification(s). Therefore, small molecules can target intracellular NP signaling pathways, increase proliferation, and direct differentiation [14,24,25]. To date, there is no report that discusses the influence of GSK-3 inhibition on expansion and differentiation of hiPSCderived NPs. Additionally, a detailed understanding of the molecular mechanisms and signaling pathways involved in the safe and efficient differentiation of hiPSCs into specific neural lineage cells is required. Therefore, in this study, we have investigated the effect of GSK-3 inhibition by the small molecule CHIR99021 on the expansion and differentiation of hiPSC-NPs in the presence and absence of growth factors, respectively. According to our data, GSK-3 inhibition increased the proliferation of NPs in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). In the absence of growth factors, GSK-3 inhibition enhanced neuronal differentiation of NPs. We have shown that GSK-3 inhibition increases the proliferation and neurogenesis of human NPs via b-catenin stabilization. Additionally, cross talk of NOTCH signaling and b-catenin are involved in enhanced proliferation of NPs. We have also shown that GSK-3 inhibition can direct hiPSCNPs differentiation into dopaminergic neurons.
Materials and Methods Human NPs expansion and differentiation hiPSC-NPs were derived and maintained as previously described [3] from a hiPSC8 line [26]. Briefly, we induced feeder-free hiPSCs that were grown for 7 days into the neural ectoderm by all-trans retinoic acid (2 mM; Sigma-Aldrich) and noggin (250 ng/mL; R&D) in the Dulbecco’s modified Eagle’s medium: Ham’s F-12 (DMEM-F12, Invitrogen, 2133120) supplemented with 5% knockout serum replacement (KOSR; Gibco, 10828-028), 1% nonessential amino acids (NEAAs; Invitrogen, 11140-035), l-glutamine (2 mM; Invitrogen, 25030-024), penicillin (100 U/mL), streptomycin (100 mg/mL; Invitrogen, 15070-063), 2% N2 (Invitrogen, 17502-048), b-mercaptoethanol (0.1 mM), and bFGF (20 ng/ mL; Royan Institute) for 6 days. Then, cells were induced for an additional 6 days in the same medium without noggin to form rosettes. Rosettes were mechanically isolated with a sterile pulled-glass pipette under a phase-contrast microscope and dissociated into single cells by trypsin/EDTA. Subsequently, dissociated cells were plated and maintained on laminin (1 mg/mL; Sigma-Aldrich, L2020) and poly-lornithine (15 mg/mL; Sigma-Aldrich, P4957)-coated tissue culture dishes in the DMEM-F12 medium supplemented with 5% KOSR, 1% NEAAs, l-glutamine (2 mM), penicillin/ streptomycin, 2% N2, b-mercaptoethanol (0.1 mM), bFGF (100 ng/mL), additional EGF (20 ng/mL; Sigma-Aldrich, E9644), and ascorbic acid (0.2 mM; Sigma-Aldrich, A8960) as
ESFANDIARI ET AL. the neural expansion medium. We changed the media every other day. Passaging was performed only at high cell densities. Passages were treated with 0.5% trypsin/EDTA (0.53 mM; Invitrogen, 25300-062) and split at 1:2–1:3 ratios. The impact of GSK-3 inhibition on proliferation of hiPSCNPs was studied in the absence (control group) and presence of the GSK-3 inhibitor, CHIR99021 (3 mM; Stemgent, 040004), for the first day after passaging. To determine the signaling pathway for proliferation in hiPSC-NPs, for 3 days we used the DAPT small molecule (1 mM; Sigma-Aldrich, D5942) to inhibit the NOTCH pathway and another small molecule, XAV939 (1 mM; Sigma-Aldrich, X3004) [27] to inhibit the b-catenin pathway. We also used pifithrin a (10 mM; Stemgent, 04-0038) to prevent apoptosis through p53 inhibition [28] (Invitrogen). Spontaneous differentiation was performed in the differentiation medium in the absence of growth factors, which included the neurobasal medium (Invitrogen, 21103-049)/DMEM-F12 (1:1), 1% B27 (Invitrogen, 17504-044), 5% KOSR, 1% N2 supplement, l-glutamine (2 mM), and penicillin/streptomycin for 2 weeks. Half of the medium was renewed every 3 days with a fresh medium that included a related small molecule.
MTT assay and PKH26 labeling To assess proliferation, we used the MTT assay, which is based on reduction of the tetrazolium salt, MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SigmaAldrich, M2128], by actively growing cells to produce a blue formazan product. MTT was diluted in the DMEM-F12 at a ratio of 1:5. We incubated the hiPSC-NPs that were in six-well plates with 500 mL of MTT solution at a temperature of 37C for at least 2 h. Next, the MTT solution was removed and 500 mL DMSO added to create a purple solution. Absorbance at 540 nm was read by an ELISA reader (Elx800; BioTek). For PKH26 labeling, hiPSC-NPs were seeded in six-well plates. After 2 h, the medium was replaced by a new medium supplemented with PKH26 (1:1,000; Sigma-Aldrich, MIN26), and the cells were incubated for 15 min. At this step, some of the plates were used as controls and analyzed by BD-FACS Calibur flow cytometer (Becton Dickinson). Subsequently, the cells were washed, a new medium without PKH26 was added, and cells were allowed to grow in the presence or absence of CHIR. After 3 days, the cells were dissociated into single cells by the application of trypsin/EDTA for 1 min. The amount of PKH26 fluorescence was detected by BDFACS Calibur flow cytometer (Becton Dickinson) and analyzed by Mod Fit software version 3.
Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction To determine gene expression at the mRNA level, we extracted total RNA by using the TRIzol reagent (Invitrogen, 15596-018). A total of 5 mg of RNA was treated with DNase I (Takara, 2270A), and 1 mg of treated RNA was used as a template for cDNA synthesis (cDNA Synthesis Kit; Fermentas, KI632), according to the manufacturer’s instructions. Synthesized cDNA (25 ng) was mixed with the 1 · Power SYBR Green PCR Master Mix (ABI, Prism, 4368702) and specific primers were added, to a final volume of 20 mL. The
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Applied Biosystems 7900 instrument was used for real-time polymerase chain reaction (PCR). Relative gene expression was analyzed by the comparative Ct method, 2 - DDCT [29]. Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd) summarizes the primer sequences that have been used in this study.
Immunoflourescence and flow cytometry analysis For immunostaining, cells were fixed by 4% paraformaldehyde (Sigma-Aldrich, P6148) for 15 min, then their membranes were permeabilized by 0.2% Triton X-100 (Sigma-Aldrich, T8532) and blocked with 10% host serum in 1% bovine serum albumin (Sigma-Aldrich, A3311). Cells were incubated at 37C with the following primary antibodies: mouse antiNESTIN (1:200; Santa Cruz, sc-71665), mouse anti-MAP2 (1:200; Sigma-Aldrich, M1406), mouse anti-GFAP (1:400; Sigma-Aldrich, G3893), and rabbit anti-PITX3 (1:200; SigmaAldrich, AV38544), which were diluted in blocking solution for 1 h. After washing 3 times with 0.1% Tween 20 (SigmaAldrich, P7949) in PBS, cells were incubated at 37C with the following secondary antibodies: goat anti-mouse FITC conjugated (1:200; Santa Cruz Biotechnology, sc-2010) and goat anti-rabbit Texas red conjugated (1:200; Santa Cruz Biotechnology, sc-2780) for 45 min. Nuclei were counterstained with DAPI (1:1,000; Sigma-Aldrich, D8417) and analyzed with a fluorescence microscope (Olympus, IX71). For flow cytometry, cells were fixed by methanol/acetone (3:7) at 4C, and then blocked with 10% serum of animal whose secondary antibody was derived for 1 h. Cells were incubated with human anti-Ki67 (1:10; ABD, HCA006) overnight at 4C. Ki67 is a marker present during all the active phases of the proliferating cell’s cycle [30]. Next, cells were incubated with goat anti-human FITC conjugated (1:200; Santa Cruz Biotechnology, SC-2456) for 1 h at room temperature. Cells directly incubated with the secondary antibody were used as controls. The FACS area flow cytometer was used for the flow cytometry analysis. Acquired data were analyzed by WinMDI software.
Western blot analysis Total proteins were extracted by the Trizol reagent, according to the manufacturer’s instructions. For each sample, 10 mg of protein was separated by 12% SDS-PAGE (90 V for 1.5 h) using the Mini-PROTEAN 3 electrophoresis cell system (Bio-Rad). Proteins were then transferred to a PVDF membrane (Bio-Rad) by the semi-dry blotting method (Bio-Rad) and the Dunn carbonate transfer buffer that consisted of NaCHO3 (10 mM), Na2CO3 (3 mM), and 20% methanol. Membranes were blocked for 1.5 h by a 2% blocking solution (Sigma-Aldrich, T8793) and then incubated overnight at 4C with the primary antibodies, mouse anti-GAPDH (1:5,000; Abcam, ab9484), anti-TH (1:10,000; Sigma-Aldrich, T1299), and rabbit anti-LMX1a (1:1,000; Abcam, ab31006). Membranes were subsequently incubated with the peroxidaseconjugated secondary antibodies, anti-mouse IgG (1:5,000; Sigma-Aldrich, A9044) and anti-rabbit IgG (1:5,000; SigmaAldrich, A9169) for 2 h at room temperature. Finally, blots were visualized by ECL detection reagents (Sigma-Aldrich, CPS-1-120). Subsequently, the films were scanned with a densitometer (GS-800; Bio-Rad).
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Statistical analysis All experiments were conducted in at least 3 independent cultures. All data were expressed as mean – standard error of mean and analyzed with one-way ANOVA followed by the Tukey’s post hoc test for multiple comparisons. P values less than 0.05 were considered significant. The results of quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) were analyzed by the independent t-test.
Results GSK-3 inhibition increased the proliferation of hiPSC-NPs In the initial step, we were interested in determining if GSK-3 inhibition by CHIR would affect proliferation in cultures of previously established hiPSC-NPs [3]. Therefore, NPs were seeded at the same density in the control and CHIR groups. Phase-contrast images showed that when cells were exposed to CHIR during the first 24 h after passaging in the presence of bFGF and EGF, an increase in proliferation was noted (Fig. 1A). However, cell death occurred when the NPs were exposed to CHIR for more than 24 h (data not shown). CHIR-treated cells reached confluency after 3 days, whereas control hiPSC-NPs became confluent after 6 days. The MTT analysis at days 1 and 3 after passaging revealed that hiPSC-NPs had higher proliferation in the presence of CHIR when compared to the control group (at least, P < 0.05, Fig. 1B). The flow cytometry analysis of Ki67-labeled cells demonstrated significantly higher Ki67-positive cells in the CHIRtreated cells compared to the control (P < 0.05, Fig. 1C). Additionally, we performed PKH26 staining to determine the dilutions of PKH26 through cell division (Fig. 1D, E). Immediately after PKH labeling, the parent generation was demarcated by mean fluorescence intensity, and the number of cellular divisions determined by decreased fluorescence in subsequent generations (Fig. 1D). Flow cytometry of labeled cells indicated that PKH26 fluorescence fell progressively with each cell division, and GSK-3 inhibition reduced the cell cycle time. There were more cells at generation 5 than control cells (at least P < 0.05, Fig. 1E). To exclude the increased cell number that was related to the inhibition of apoptosis, we used pifithrin a. This is a small molecule that inhibits p53, therefore preventing apoptosis. According to the MTT analysis, there was no significant difference between pifithrin a and the control (Fig. 1F). Therefore, the increased cell numbers were related to cell proliferation, not reduction by cell death. To assess the long-term passage effect of CHIR on hiPSCNPs, we continued to expose the cells to CHIR during the first day after culture for a total of 10 passages. The cells in the CHIR group were passaged every 3 days, whereas those in the control group were passaged every 6 days. The MTT assay of CHIR-treated cells across 10 passages showed more proliferation for CHIR-treated cells compared to the control group (P < 0.0001, Fig. 2A). The gene expression analysis for the NP genes, NESTIN, SOX1, and PAX6 after 10 passages showed increased expressions of all 3 genes in the CHIRtreated group. Decreased expression of GFAP was noted in the CHIR-treated cells (Fig. 2B). To assess the differentiation potential of CHIR-treated hiPSC-NPs, cells were differentiated at passage 10 by growth
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FIG. 1. GSK-3 inhibition in the proliferation of hiPSC-NPs. (A) Phase-contrast microscopy of hiPSC-NPs at 24 and 72 h after GSK-3 inhibition. (B) The MTT assay for quantification of the proliferation in hiPSC-NPs upon GSK-3 inhibition at days, 0, 1, and 3 in control and CHIR-treated cells. Treatment of the cells by CHIR increased MTT absorbance. (C) Flow cytometry for Ki67-positive cells, a marker for cell division that expresses in the nucleus of proliferating cells, causes an increase in cell proliferation in the presence of CHIR. (D) The flow cytometry analysis of the generations in the control group and its comparison with CHIR-treated cells using PKH26 staining. (E) Statistical presentation of the data for parent, G4, and G5 in the CHIR group compared to the control group. The number of cells in G5 was more than the control at this stage. (F) The MTT assay showed that pifithrin a, a small molecule that prevents apoptosis by inhibiting p53, could not increase cell numbers compared to the control group. Therefore, enhancement in cell numbers was related to cell proliferation, not to reductions in cell death. P, parent; G, generation. Independent t-test, aP < 0.05, bP < 0.01. hiPSC-NPs, human-induced pluripotent stem cell-derived neural progenitors; GSK-3, glycogen synthase kinase 3; CHIR, CHIR99021. factor withdrawal. After 14 days, hiPSC-NPs of the control group spontaneously differentiated into neurons and astrocytes, a property consistent with normal multipotent NPs (Fig. 2C). Additionally, hiPSC-NPs that received CHIR for 10 passages were CHIR-dependent for their differentiation. These cells produced more neurons rather than glial cells at a higher efficiency when compared to the control group (Fig. 2C). The real-time RT-PCR analysis of spontaneously differentiated NPs indicated that expressions of GFAP, MAP2, NEUROD1, and TH influenced significantly in the presence of CHIR in the differentiation medium compared with the control group (no CHIR during differentiation). However, the potential of differentiated cells that were passaged 0 or 10
times in the presence of CHIR were not significantly different (Fig. 2D).
Possible pathway of hiPSC-NPs proliferation upon GSK-3 inhibition GSK-3 is a multifunctional protein that regulates cross talk with different pathways such as b-catenin and NOTCH signaling. GSK-3 is known to have an inhibitory effect on NOTCH signaling. Phosphorylation of threonine 2068, 2070, or 2074 in the ankyrin repeats at the c-terminal of NOTCH by GSK-3 inhibits transcription of the Cross-talk of GSK-3 [31–34]. GSK-3 phosphorylates b-catenin that is to be marked for degradation [35,36].
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FIG. 2. Characterization of hiPSC-NPs proliferation and differentiation capacity upon CHIR treatment. (A) The MTT assay for determination of proliferation capacity in the CHIR group compared to the control during 10 passages (Regression, P < 0.0001). The application of CHIR increased cell numbers. (B) The qRT-PCR analysis for CHIR-treated hiPSC-NPs for 10 passages compared to the control. In presence of CHIR, the NPs expressed more SOX1 and PAX6, stemness markers and less GFAP, a differentiation marker, compared to the control group. However, the dopaminergic fate of NPs did not change during 10 passages, as shown by the expression of LMX1A, which did not change significantly. (C) hiPSC-NPs treated during 10 passages by CHIR maintained the potential of spontaneous differentiation into neurons (MAP2 + ) and glials (GFAP + ) as detected by immunofluorescence staining. However, their differentiation into neurons increased, whereas differentiation into glials decreased when CHIR was added to the medium during spontaneous differentiation of hiPSC-NPs that had received CHIR for 10 passages. (D) The gene expression analysis by qRT-PCR for spontaneous differentiation of NPs showed that expressions of MAP2, NEUROD1, and TH upregulated, with downregulation of GFAP in the presence of CHIR during differentiation compared to the control (no CHIR during differentiation). However, there were no significant differences in genes from the neural progenitor (NP) derivatives that were passaged 0 or 10 times in the presence of CHIR. b: p < 0.01, c: p < 0.001. qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction. Cross-talk of GSK-3 inhibition of b-catenin and NOTCH signaling in the proliferation of NPs was checked by XAV939 (a b-catenin inhibitor) and DAPT (a NOTCH-signaling inhibitor) in the NP medium for 3 days. The MTT assay showed that, after 3 days, addition of CHIR did not compensate cell expansion in the presence of XAV939 and DAPT when compared with the control group (at least P < 0.05, Fig. 3A). It was expected that CHIR might control the b-catenin and NOTCH-signaling target genes during the first 24 h of GSK-3 inhibition. Expression data indicated that mRNA of the CMYC and CYCLIND1 genes (target genes of b-catenin) [37] increased 12 h after GSK-3 inhibition (P < 0.05 for C-MYC, P < 0.01 for CYCLIND1, Fig. 3B). Inhibition of b-catenin by XAV significantly blocked their expressions compared to CHIR (P < 0.01, Fig. 3B). No changes were detected in the expressions of C-MYC and CYCLIND1 24 h after GSK-3 inhibition (Fig. 3B).
The gene expression analysis for NOTCH target genes showed that 30 min after CHIR treatment, expression of HES5 became elevated and continued until 1 h after GSK-3 inhibition (P < 0.05, Fig. 3C). However, its expression decreased upon further treatment with CHIR. When the cells inhibited for NOTCH (by DAPT) and GSK-3 (by CHIR), expression of HES5 decreased at 30 min (P < 0.01), 1 h (P < 0.05), 12 and 24 h (at least P < 0.05) after treatment. The expression of HES1 increased after GSK-3 inhibition (P < 0.05). NOTCH inhibition did not decrease HES1 expression when both NOTCH and GSK-3 were inhibited; however, when NOTCH was exclusively inhibited its expression remained constant. HES1 expression decreased at 12 and 24 h (at least P < 0.05) after treatment. These findings showed that GSK-3 inhibition promoted hiPSC-NP proliferation through the activation of the b-catenin and NOTCHsignaling pathways.
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FIG. 3. GSK-3 inhibition and its cross-talk with NOTCH and b-catenin signaling. (A) The MTT assay for the inhibition of NOTCH and bcatenin and their impact on the proliferation of hNPs. Cell proliferation reduced in the presence of XAV (b-catenin inhibitor) and DAPT (NOTCH inhibitor). With CHIR, it was demonstrated that CHIR influenced cell proliferation by increasing b-catenin and NOTCH signaling. (B) The gene expression analysis for b-catenin target genes C-MYC and CYCLIND1 at 12 and 24 h after GSK-3 inhibition in the CHIR group. Expressions of C-MYC and CYCLIND1 increased 12 h after CHIR treatment; however, the application of XAV reduced their expressions at the same time. (C) The gene expression analysis for NOTCH target genes, HES5 and HES1, at 30 min, 1 h, 90 min, 12 and 24 h after GSK-3 inhibition in the CHIR group. The application of CHIR increased the expressions of HES5 and HES1, at 30 and 90 min, respectively, whereas addition of DAPT reduced the related genes. One-way ANOVA followed by the Tukey’s post hoc test for multiple comparisons. aP < 0.05, bP < 0.01, and cP < 0.001. In each experiment, the CHIR, DAPT, and XAV groups were compared to the control. The combined groups, CHIR + DAPT and CHIR + XAV, were compared to CHIR.
Possible pathway for hiPSC-NPs differentiation upon GSK-3 inhibition hiPSC-NPs were allowed to differentiate for 14 days in the absence of bFGF and EGF, but with the addition of CHIR during this time. The expressions of MAP2, GFAP, NEUROD1, and NGN3 were measured by qRT-PCR and/or immunofluorescence staining (Figs. 2D and 4). In the presence of CHIR, the number of MAP2-positive cells increased (90%) compared to the control (29%; P < 0.01, Fig. 4A, B), while the number of GFAP-positive cells decreased (7%) compared with the control (62%; P < 0.05, Fig. 4A, B). The qRT-PCR analysis showed a significantly increased expression of MAP2, whereas the astrocyte marker, GFAP, decreased upon differentiation (P < 0.001, Fig. 2D). Additionally, expression of the b-catenin target genes, NGN3 and NEUROD1, that were implicated in neurogenesis expressed significantly after CHIR treatment (P < 0.01 Fig. 2D, data for NG3 not shown). To explore the potential mechanisms of neurogenesis by GSK-3 inhibition, DAPT and XAV were applied for NOTCH and b-catenin inhibition, respectively. As with the proliferation assay, hiPSC-NPs were differentiated in 6 different groups. In the DAPT- and CHIR + DAPT-treated cells, expressions of MAP2 and GFAP did not significantly change during differentiation compared to the CHIR group (Fig. 4A, B). The application of XAV completely blocked neurogenesis as no MAP2-positive cells were detected (P < 0.01). The application of CHIR + XAV did not improve neurogenesis (Fig. 4A, B), while NPs continued their glial differentiation by 71% in the presence of XAV or XAV + CHIR (P < 0.05, Fig. 4A, B). Our results showed that GSK-3 inhibition by CHIR increased neurogenesis through b-catenin stabilization, independent of NOTCH signaling. Evaluation of cell proliferation by the MTT assay indicated that when hiPSC-NPs were treated with CHIR for differen-
tiation, the total number of differentiated cells remained unchanged compared to the control group (Fig. 4C). The application of another GSK-3 inhibitor, SB216763, led to enhanced neurogenesis and reduced giologenesis (Fig. 4D).
GSK-3 inhibition may bias cells toward a dopaminergic fate To investigate if the Wnt-signaling pathway can modulate DA neuronal development, we treated hiPSC-NPs with CHIR during differentiation. Expressions of LMX1A and LMX1B, both DA precursor markers, significantly changed at day 7, but their expressions downregulated upon maturation. Expressions of the mature dopaminergic neuron markers TH, NURR1, EN1, and NGN2 were upregulated (at least P < 0.05) at day 14 postinitiation of spontaneous differentiation in the presence of CHIR (Fig. 5A). To further assess the dopaminergic fate of differentiating cells, we performed immunostaining and western blot. Although a few PITX3-positive cells ( < 1%), which are a specific transcription factor for dopaminergic neurons [38], have been observed in the control group, the addition of CHIR to differentiating NP cultures increased the number of double-positive MAP2/PITX3 cells to 98% (Fig. 5B). These cells were also THpositive (Fig. 5B). Interestingly, we found that treatment of differentiating precursors with CHIR resulted in upregulation of the TH protein level as detected by western blot (Fig. 5C, D).
Discussion The proliferation and differentiation of hiPSC-NPs into a definitive neuronal subtype, such as DA neurons, is of great interest because of their potential use in regenerative medicine to treat disorders such as Parkinson’s disease. Understanding the signaling events during hiPSC-NPs
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FIG. 4. b-catenin and NOTCH signaling and their cross-talk in the neurogenesis of hiPSC-hNPs. (A) MAP2 and GFAP immunostaining in the suppressed NOTCH (DAPT) versus suppressed b-catenin (XAV) and CHIR groups. (B) Quantification of immunofluorescence images for MAP2 and GFAP. The addition of XAV suppressed MAP2, but induced GFAP expression in the presence of CHIR, while DAPT did not significantly change MAP2 and GFAP expressions. (C) The MTT assay showed that when hiPSC-NPs were treated with CHIR for differentiation, the total number of differentiated cells remained unchanged. (D) Immunostaining for MAP2 and GFAP after application of another GSK-3 inhibitor, SB216763. A similar trend was observed for this GSK-3 inhibitor as enhanced neurogenesis and reduced gliogenesis (independent t-test, aP < 0.05, bP < 0.01). Color images available online at www .liebertpub.com/scd
proliferation and differentiation is essential for the generation of more efficient protocols with which to establish desired neurons from hiPSC-NPs. There are many reports that have shown the importance of Wnt signaling for different stages of proliferation and differentiation. The role of Wnt signaling (which results in the inhibition of GSK-3), however, in the proliferation and differentiation of hiPSC-NPs has not yet been reported. In this study, we have found that GSK-3 inhibition by small molecule CHIR greatly promoted proliferation of hiPSC-NPs in the presence of bFGF and EGF, and improved neuronal and DA subtype differentiation of hiPSC-NPs. However, key issues such as production, cellpermeability, reversibility of inhibition, and specificity of binding aimed at overcoming safety concerns should be addressed before using small molecules to generate cells for regenerative medicine.
GSK-3 inhibition increased the proliferation of hiPSC-NPs through activation of b-catenin and NOTCH signaling GSK-3 inhibition plays an important role in adult stem/ progenitor cells and in neural development [39–45]. Continuous activation of GSK-3 has been found in the brains of
patients with Alzheimer disease [46] and psychiatric disorders [9]. It has also been demonstrated that NPs of mice with a GSK-3 deletion show hyperproliferation [43]. Therefore, GSK-3 has attracted a substantial amount of attention for developmental biology and therapy. Here, we found that GSK-3 inhibition by CHIR (3 mM) during the first day after passaging increased the proliferation of hiPSC-NPs in the presence of bFGF and EGF. Our findings on enhanced proliferation of hiPSC-NPs agreed with recent in vitro studies, which demonstrated enhanced influence of SB-216763 [14,25] or R3303544 [4o7] GSK-3 inhibitors on the proliferation of the hNPs cell line ReNcell VM or murine subventricular zone-derived NPs. In contrast, it has been demonstrated that the GSK-3 inhibitor SB-216763 did not significantly affect proliferation in murine neonatal NPs [48,49]. IM-12 decreased cell growth of the human NP line, ReNcell VM [17]. Additionally, it has been shown that inhibition of GSK-3 by 6-bromoindirubin-3¢oxime or CHIR99021 reduced the proliferation of adult human olfactory epithelium NPs [50]. Differing results may be a result of different GSK-3 inhibitors, the origin of the NPs, or cross-talk between different ligands such as bFGF and EGF in the medium [13,45]. Here, we have used bFGF along with CHIR during maintenance of NPs. bFGF increases NPs
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FIG. 5. GSK-3 inhibition and neuronal differentiation toward dopaminergic neurons. (A) Relative expressions of LMX1A and LMX1B on day 7 of differentiation and dopaminergic genes TH, NGN2, NURR1, EN1, and MSX1 on day 14 after differentiation. Dopaminergic precursors generated at day 7, with cellular differentiation toward mature dopaminergic neurons at day 14. (B) PITX3 and TH immunostaining for differentiated neurons treated with CHIR compared with the control cells at day 14. (C) The western blot analysis of differentiated cells at day 14 for expressions of TH and LMX1a at the protein level with the GAPDH protein as the internal control. (D) Quantification of band intensities from (C). These data demonstrated CHIR treatment of differentiating NPs might lead to dopaminergic neurons (independent t-test, aP < 0.05, bP < 0.01, cP < 0.001). Color images available online at www .liebertpub.com/scd
proliferation and concurrently inhibits their differentiation. It has been reported that bFGF signaling through phosphatidylinositol 3 kinase (PI3K) activation inactivates GSK-3, resulting in the accumulation of b-catenin in a manner different from that in the Wnt canonical pathway [14]. To explore the mechanisms by which GSK-3 inhibition increased proliferation, we used XAV to inhibit b-catenin signaling and DAPT to inhibit NOTCH signaling. We found that the impact of CHIR on hiPSC-NPs proliferation reversed in the presence of XAV and DAPT. These results showed that inhibition of GSK-3 at this stage resulted in b-catenin stabilization and NOTCH signaling. The downregulation of CMYC and CYCLIND1 12 h after GSK-3 inhibition, and HES5 and HES1 induction after 30 and 90 min, respectively, confirmed cross-talks among GSK-3, b-catenin, and NOTCHsignaling pathways in the proliferation of NPs. It has been previously described that ventral midbrain precursors can respond to GSK-3 inhibitors, kenpaullone, and indirubine-3-monoxime (I3M), by stabilization of bcatenin [51]. Accumulation of b-catenin in the nucleus resulted in proliferation by activating LEF/TCF transcription factors and concurrent repression of neuronal differentiation by potentiating the NOTCH1-RBP-Jkappa-signaling pathway [14,34,48]. b-catenin and the NOTCH1 intracellular domain make a molecular complex with the promoter region of the antineurogenic Hes1 and Hes6 genes, permitting their expression [14]. Also, it has been demonstrated that lithium chloride treatment or Wnt-1 overexpression resulting in GSK-3 inhibition leads to the upregulation of the Hes1 promoter [34]. This signaling interplay is particularly essential for NPs expansion, because misexpression of dominantactive GSK-3b completely suppresses the NP self-renewal, increasing their differentiation into neurons. Thus, the GSK-
3b/b-catenin signaling axis is regulated by FGF and Wnt signals by linking the cell proliferation to the inhibition of differentiation [14].
GSK-3 inhibition increased neurogenesis independent of NOTCH signaling This study demonstrated that the inhibition of GSK-3 enhanced neuronal differentiation and blocked gliogenesis of hiPSC-NPs in the absence of bFGF and EGF to 90% MAP2positive cells. It has been observed that Wnt signaling increased neuronal differentiation of mouse cortical NPs [45,52]. Similarly, GSK-3 inhibition in cultures of embryonic rat ventral mesencephalon cells led to increased neuronal differentiation [51], differentiated adult rat hippocampusderived neurospheres in vitro [49], and increased neurogenesis of the hNPs cell line ReNcell VM up to 5.7% compared to 2.6% in control cells [25]. Also, GSK-3 inhibitors promoted neurite outgrowth [53]. Strong knockdown of GSK-3 significantly decreased axonal growth in dissociated neuronal cultures [54]. In our study there was decreased expression of GFAP, an astrocyte marker. These data agreed with a recent report on neurospheres derived from neonatal mouse forebrains [55]. The exact mechanism by which GSK-3 inhibitors increased neurogenesis and their effect on the fate of neurons is unknown. To explore the cellular mechanism by which GSK-3 inhibition promotes neurogenesis, we have inhibited b-catenin and NOTCH signaling. Immunostaining data revealed that GSK-3 or NOTCH inhibition increased neurogenesis. Treatment of cells with both GSK-3 and NOTCH inhibitors also showed similar results. Therefore, neurogenesis occurred independent of NOTCH signaling.
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However, the application of XAV suppressed neurogenesis and induced gliogenesis, which indicated the importance of b-catenin in this regard. It has been demonstrated that b-catenin stabilization by CHIR increases expression of the proneuronal genes, NEUEOD1 and NGN3 [45,56]. NeuroD1 is essential for CNS development, particularly in the hippocampus and cerebellum [57,58]. Overexpression of NeuroD1 is sufficient to induce neuronal differentiation of adult hippocampal neurons [59]. Ngn3 expression in rodents regulates glial differentiation of spinal cord glial precursors [60] and promotes early neurogenesis of the chick spinal cord [37].
Therefore, a promising strategy to increase neuronal yield in hiPSC-NP cultures would consist of a 2-step process that inhibits GSK-3. Initially, hiPSC-NPs would be expanded in conditions that maximize their proliferation by using the expansion medium in the presence of bFGF and EGF. In the second culture step, they would be induced toward neuronal lineage and maturation in the presence of a GSK-3 inhibitor in the differentiation medium in the absence of bFGF and EGF. Our strategy may improve the application of pluripotent stem cell-derived NPs and neuronal cells for cell replacement therapy in neurological disorders such as Parkinson’s disease.
GSK-3 inhibition may change the propensity of differentiating NPs toward a dopaminergic fate
Acknowledgments
It has been reported that GSK-3b inhibitors, I3M and kenpaullone, stabilize b-catenin, and that overexpression of b-catenin in ventral mesencephalic precursors results in increased DA differentiation [51]. Additionally, Wnts are key regulators of proliferation and differentiation of DA precursors during ventral midbrain neurogenesis [61]. In this regard, we have found that GSK-3 inhibition directs neuronal differentiation of hiPSC-NPs to a dopaminergic fate. Also, we have detected DA precursors at day 7 of differentiation due to their high expressions of LMX1A and LMX1B. Immunostaining for PITX3 as a specific transcription factor for midbrain dopaminergic neurons has shown the presence of *90% PITX3-positive cells in hiPSC-NPs that differentiated upon GSK-3 inhibition. Additionally, TH expression at the protein level and mRNA expressions of TH, NURR, MSX1, EN1, and NGN2 increased in the CHIR group compared to the control group at day 14 after differentiation. It has been reported that the Wnt/b-catenin pathway inhibitor Dickkopf1 (Dkk1) increased both neuroectodermal differentiation and the number of mouse ESC-derived DA neurons [44]. Recent studies have also demonstrated that iPSC-NPs can be differentiated in cultures to generate neurons, which produce significant functional improvement when transplanted in rodent models of Parkinson’s disease [62,63], monkey spinal cord injury [64], and damaged swine neural retina [65]. More recently, the Studer’s group have shown that midbrain floor-plate precursors and DA neurons could be derived from embryonic stem cells days after exposure to small molecule activators of sonic hedgehog and canonical Wnt signaling [66]. Additionally, long-term engraftment in Parkinsonian mice, rats, and monkey models indicate robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior, and improvements in tests of forelimb use and akinesia [66].
Conclusions Taken together, our data indicated that b-catenin stabilization by GSK-3 inhibition increased proliferation of hiPSCNPs by increasing the expression of cell cycle-promoting genes such as C-MYC and CYCLIND1. Neurogenesis was promoted by increased expressions of proneural genes such as NEUROD1 and NGN3. Additionally, in the presence of CHIR in the differentiation medium, the neuronal differentiating cells showed a bias toward a dopaminergic fate.
This study was funded by grants provided from the Royan Institute and the Iranian Council of Stem Cell Research and Technology.
Author Disclosure Statement The authors declare that they have no competing financial interests.
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Address correspondence to: Dr. Hossein Baharvand Department of Stem Cells and Developmental Biology Cell Science Research Center Royan Institute for Stem Cell Biology and Technology ACECR, P.O. Box 19395-4644 Tehran Iran E-mail:
[email protected] Received for publication December 5, 2011 Accepted after revision May 29, 2012 Prepublished on Liebert Instant Online May 29, 2012
