Human cytomegalovirus induces apoptosis in neural stem/progenitor cells derived from induced pluripotent stem cells by generating mitochondrial dysfunction and endoplasmic reticulum stress
© Nakamura et al.; licensee BioMed Central Ltd. 2013
Received: 17 April 2013
Accepted: 15 October 2013
Published: 21 October 2013
Congenital human cytomegalovirus (HCMV) infection, a leading cause of birth defects, is most often manifested as neurological disorders. The pathogenesis of HCMV-induced neurological disorders is, however, largely unresolved, primarily because of limited availability of model systems to analyze the effects of HCMV infection on neural cells.
An induced pluripotent stem cell (iPSC) line was established from the human fibroblast line MRC5 by introducing the Yamanaka’s four factors and then induced to differentiate into neural stem/progenitor cells (NSPCs) by dual inhibition of the SMAD signaling pathway using Noggin and SB-431542.
iPSC-derived NSPCs (NSPC/iPSCs) were susceptible to HCMV infection and allowed the expression of both early and late viral gene products. HCMV-infected NSPC/iPSCs underwent apoptosis with the activation of caspase-3 and −9 as well as positive staining by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Cytochrome c release from mitochondria to cytosol was observed in these cells, indicating the involvement of mitochondrial dysfunction in their apoptosis. In addition, phosphorylation of proteins involved in the unfolded protein response (UPR), such as PKR-like eukaryotic initiation factor 2a kinase (PERK), c-Jun NH2-terminal kinase (JNK), inositol-requiring enzyme 1 (IRE1), and the alpha subunit of eukaryotic initiation factor 2 (eIF2α) was observed in HCMV-infected NSPC/iPSCs. These results, coupled with the finding of increased expression of mRNA encoding the C/EBP-homologous protein (CHOP) and the detection of a spliced form of X-box binding protein 1 (XBP1) mRNA, suggest that endoplasmic reticulum (ER) stress is also involved in HCMV-induced apoptosis of these cells.
iPSC-derived NSPCs are thought to be a useful model to study HCMV neuropathogenesis and to analyze the mechanisms of HCMV-induced apoptosis in neural cells.
KeywordsHuman cytomegalovirus iPS cells Neural stem/progenitor cells Apoptosis ER stress
Congenital cytomegalovirus (CMV) infection is a major cause of birth defects resulting mainly from primary CMV infection during pregnancy. At birth, approximately 5 to 10% of congenitally infected newborns are estimated to be symptomatic exhibiting multi-organ disorders including neurological defects such as mental retardation, sensorineural hearing loss, and microencephaly [1, 2]. A latest study showed that if laboratory findings including those from magnetic resonance imaging (MRI) images of the brain are considered, up to 30% of congenitally infected newborns exhibit some abnormal signs . Sixty to 90% of congenitally infected children who are symptomatic at birth, and 10 to 15% of those who are asymptomatic at birth develop one or more long-term sequelae. Although CMV infects a wide variety of cell types, infection of the nervous system gives most serious and long-lasting damages to the host.
As a part of understanding the HCMV neuropathogenesis, it is important to scrutinize the cellular response to CMV infection in neural cells. Some human neural cell lines can be infected with HCMV with different permissiveness to HCMV gene expression and replication [4–7]. A recent study has shown that neural progenitor cells isolated from developing human brain tissues are susceptible to CMV infection and undergo apoptosis following infection [8, 9]. However, the amount of neural cells obtainable from human brain tissues is limited.
Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are characterized by the ability to differentiate into tissues derived from any of the three embryonic germ layers. Recent advances in the method to induce efficient differentiation of either ESCs or iPSCs into specific cell lineages offer an opportunity to establish model systems for viral infections of various cell types, including neural cells. Furthermore, differentiated cells derived from pluripotent stem cells are obtainable in potentially unlimited amounts. Previous works revealed that while mouse ESCs are not susceptible to murine CMV (MCMV), NSPCs that are differentiated from them are susceptible and their proliferation and differentiation are suppressed by MCMV [10–13]. Experiments with human ESCs are, however, complicated with ethical problems.
In this study, to analyze the pathological effects of HCMV on neural cells, we prepared NSPCs from human iPSCs and examined whether NSPCs are susceptible to HCMV infection. The results indicated that NSPCs are susceptible to HCMV infection and undergo apoptosis caused by mitochondrial dysfunction and endoplasmic reticulum (ER) stress.
Cells and viruses
The human fetal lung fibroblast MRC5 was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). The human foreskin fibroblast cell line hTERT-BJ1 immortalized with the human telomerase reverse transcriptase (Clontech, Palo Alto, CA) was grown in a medium consisting of 4 parts of DMEM and 1 part of medium 199 (Sigma) supplemented with 10% FBS, 1 mM sodium pyruvate (Sigma), and 2 mM glutamine (Invitrogen). HCMV laboratory strain Towne (ATCC VR-977) was propagated in hTERT-BJ1 cells. The human iPSC line MRC-iPS-25 that was established from MRC5 by retroviral vector-mediated transduction of the c-Myc, Oct-4, Klf4, and Sox2 genes [14, 15] were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs) in an iPSC medium consisting of Knockout DMEM/F12 (Invitrogen) supplemented with non-essential amino acids (0.1 mM, Invitrogen), glutamax I (1 mM, Invitrogen), 20% Knockout Serum Replacement (Invitrogen), β-mercaptoethanol (55 μM, Invitrogen) and basic fibroblast growth factor (10 ng/mL; Peprotech, Rocky Hill, NJ).
Induced differentiation on iPSCs into neural stem cells
MRC-iPSC-25 cells cultured under feeder-free conditions were induced to differentiate into neural stem/progenitor cells (NSPCs) by the method of dual inhibition of the SMAD signaling pathway described previously . In brief, feeder-free iPSCs were treated with the mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) containing Y27632 (Wako Pure Chemicals, Osaka, Japan) and maintained with a daily medium change for 4 days. Then the medium was replaced with iPSC medium supplemented with SB431542 (10 nM, Wako Pure Chemicals) and Noggin (500 ng/ml, Wako Pure Chemicals). This date was designated day 0. On day 2, culture medium was replaced with a medium consisting of 3 parts of iPSC medium and 1 part of N2 medium (Knockout DMEM/F12 containing 1× N2 supplement) supplemented with SB431542 (10 nM) and Noggin (500 ng/ml). On day 4, culture medium was replaced with a medium consisting of 1 part of iPSC medium and 1 part of N2 medium supplemented with SB431542 (10 nM) and Noggin (500 ng/ml). On day 6, cells were expanded in StemPro NSC SFM (Invitrogen). MRC-iPSC-25 cells cultured under feeder-free conditions and NSPC/iPSCs were infected with the Towne strain HCMV at a multiplicity of infection (MOI) of 1 plaque forming unit (PFU) per cell. To detect infectious virions produced from HCMV-infected NSPC/iPSCs, supernatant was collected and replaced with fresh medium every two days after infection. hTERT-BJ1 cells were inoculated with the supernatant and examined by IFA for expression of IE1/IE2.
Antibodies used were as follows: rabbit anti-Sox2, rabbit anti-Nanog, rabbit anti-Oct-4, rabbit anti-cleaved caspase-3, rabbit anti-cleaved caspase-9, rabbit anti-phospho-eIF2α (Ser51), rabbit anti-phospho-PERK (Thr980), and rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185)(Cell Signaling Technology, Beverly, MA); mouse anti-CMV IE1/IE2, rabbit anti-Musashi-1, and rabbit anti-cytochrome c (Millipore, Temecula, CA); rabbit anti-Nestin and mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO); rabbit anti-Pax6 (Covance, Princeton, NJ), mouse anti-CMV gB (Abcam, Cambridge, MA); mouse anti-pp65 (Virusys Corporation, Sykesville, MD); rabbit anti-phosphorylated IRE1α (Pierce/Thermo Scientific, Rockford, IL); Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR); horseradish peroxidase-conjugated donkey anti-rabbit IgG and horseradish peroxidase-conjugated sheep anti-mouse IgG (GE Healthcare, UK).
Immunofluorescence microscopy and immunoblot analysis
Cells were fixed with 4% parafolmaldehyde in PBS (Wako chemicals) at room temperature (RT) for 15 min. After fixation, cells were treated with 1.0% Triton X-100 in PBS for 15 min at RT and blocked with 10% goat serum in PBS for 30 min. Cells were incubated with the primary antibody at 4°C overnight, followed by washing in PBS and incubation with the corresponding secondary antibody at 37°C for 1 h. Nuclei were stained with DAPI. For the assessment of cell death, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was performed according to the manufacturer’s instructions (Roche). Immunoblot analyses were performed as described previously .
Reverse transcriptase (RT)-PCR and real-time quantitative RT-PCR
List of primer sequences for RT-PCR
Preparation of human iPSC-derived neural stem/progenitor cells
NSPC/iPSCs prepared by induced differentiation of MRC-iPS-25 cells proliferated in a monolayer and displayed a rounded, immature neural morphology (Figure 1A). IFA (Figure 1C) showed that NSPC/iPSCs expressed the NSC markers Nestin, Sox2, and Pax6, indicating that NSPC/iPSCs have the authentic NSPC phenotype.
In vitro HCMV infection of iPSC-derived NSPCs
Expression of HCMV genes in NSPC/iPSCs was evaluated at the protein level by immunoblot analysis on day 1, 2, 5, and 7 following HCMV infection. As shown in Figure 2D, the immediate-early protein IE1 was first detected at 1 dpi and its level increased until 5 dpi. Another immediate-early protein IE2 was detected a little later, becoming visible at 5 dpi. The expression of the HCMV lower matrix protein pp65 (ppUL83), already visible at 1 dpi, was markedly elevated at 5 and 7 dpi. The HCMV envelope glycoprotein B (gB; UL55) was detected at 5 to 7 dpi. Thus the expression of HCMV proteins of both immediate-early and late functions was demonstrated in NSPC/iPSCs.
We next examined the expression of cellular mRNAs encoding the pluripotency and neural differentiation markers (Figure 2E). Expression of the iPSC markers Nanog and Oct-4 remained at low levels following infection with HCMV, although that of Nanog tapered. While expression of the NSPC markers Sox2 and Pax6 were kept at high levels following HCMV infection, that of another NSPC marker Nestin was markedly suppressed at 7 dpi. In addition, expression of the neuronal marker microtubule-associated protein 2 (MAP2), the astrocyte marker glial fibrillary acidic protein (GFAP), and the oligodendrocyte marker oligodendrocyte-specific protein (OSP) was detected at low levels. Interestingly, Sox1, a marker specific to the neuroectodermal lineages , was markedly upregulated following infection with HCMV. Expression of the NSPC markers was evaluated also at the protein level by immunoblot analysis on 1, 2, 5, and 7 dpi (Figure 2F). In accordance with the results with RT-PCR, expression of Pax6 and Nestin was confirmed, and that of Nestin was found markedly decreased 7 dpi. Another NSPC marker Musashi-1 was also detected. To examine whether HCMV-infected NSPC/iPSCs produce infectious virions, culture supernatants collected from Towne HCMV-infected NSPC/iPSCs were inoculated to hTERT-BJ1 cells. Inoculated cells expressed IE1/IE2 indicating that infectious virions were produced from HCMV-infected NSPC/iPSCs (Figure 2G). The supernatant contained 30 PFU/mL of HCMV at 4, 6, 8 dpi, while no plaque forming virus was detected at 2 dpi.
HCMV infection induces apoptosis in iPSC-derived NSPCs
Unfolded protein response in HCMV-infected NSPC/iPSCs
Important findings in this study are as follows: i) NSPC/iPSCs derived from MRC-iPS-25 cells were susceptible to HCMV infection and allow the expression of viral gene products of both early and late functions and production of infectious virions. In contrast, MRC-iPS-25 cells before induction of differentiation was either resistant to HCMV or did not support the expression of HCMV immediate-early genes; ii) the HCMV-infected NSPCs undergo apoptosis; and iii) the mechanism of the apoptosis included cytochrome c release from mitochondria to cytosol and activation of UPR-related signaling pathways.
Neuropathogenesis of HCMV infection has been studied mainly with neural cells isolated from human brain. These studies demonstrated that HCMV can infect human neural precursor cells (NPCs) isolated from fetal brains and interfere with their differentiation. Luo et al.  showed that HCMV infection in primary NPCs reduced the expression of Nestin, suggesting that HCMV affects the differentiation potential of NPCs. Similar results were also obtained from experiments with mouse NSCs infected with MCMV [10, 13, 24]. Those previous findings obtained from experiments with primary cultures of brain-derived neural cells were thus mostly reproduced in our experiments using NSPC/iPSCs. In addition, similar to the results of Odeberg et al.  that used NPCs derived from human brain, we also demonstrated that HCMV infection induced apoptosis in NSPC/iPSCs obtained from iPSCs. It is thus supposed that neural cells differentiated from iPSCs are a useful model to investigate neural pathogenesis of HCMV. In the human brain, NSCs are predominantly found in the subventricular region where CMV infections preferentially occur [25, 26]. Analysis on the effects of HCMV infection on NSPCs can be therefore particularly relevant.
In the regulation of cellular apoptotic responses, mitochondrial dysfunction and ER stress are involved in the activation of the initiator caspase caspase-9 that functions as a trigger of cascade protease reactions leading to cell death. The finding of cytochrome c release from mitochondria to cytoplasm in HCMV-infected NSPC/iPSCs indicates that mitochondrial dysfunction is involved in the activation of caspase-9 in these cells. In addition, the demonstration of phosphorylated forms of proteins involved in UPR, including PERK, JNK, IRE1α, eIF2α, as well as that of unconventional splicing of XBP1 mRNA and up-regulation of CHOP, indicate that ER stress also plays a role in HCMV-induced apoptosis of NSPC/iPSCs. These results are in accordance with the work reported by Isler et al.  who demonstrated that HCMV-induced UPR in human foreskin fibroblasts. HCMV is known to encode anti-apoptotic proteins such as viral inhibitor of caspase-8-induced apoptosis (vICA) encoded by UL36 , and pUL38 which protects against ER stress-induced cell death by modulating the UPR pathway . Our RT-PCR analysis demonstrated that such viral anti-apoptotic genes were expressed at transcription level in NSPC/iPSCs following HCMV infection (Figure 2B). Although these viral anti-apoptotic proteins did not block apoptosis of NSPC/iPSCs, they might have contributed for efficient viral replication by delaying apoptosis.
iPSCs are expected to be an innovative tool for not only regenerative medicine but also for the elucidation of pathogenesis of various diseases. Recent studies have shown that human iPSCs can be used also for modeling viral infection. Hepatocyte-like cells derived from human iPSCs were shown to be susceptible to hepatitis virus C infection and supported its replication [30, 31]. Sensory neurons derived from human iPSCs were reported to be susceptible to infection with both varicella-zoster virus and herpes simplex virus . While the present work was in progress, D’Aiuto and others reported on the preparation of an iPSC-derived model of HCMV infection in neural precursor cells . Whereas our data described in the present study is largely consistent with their results, we further analyzed the mechanisms of apoptosis induction and elucidated the involvement of mitochondrial dysfunction and ER stress.
In conclusion, human NSPCs derived from iPSCs can be a useful model to study HCMV neuropathogenesis associated with congenital HCMV infection. They can be particularly valuable in analyzing the mechanisms of HCMV-induced apoptosis in neural cells.
Induced pluripotent stem cell
Embryonic stem cell
Neural stem/progenitor cell
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
Unfolded protein response
PKR-like eukaryotic initiation factor 2a kinase
c-Jun NH2-terminal kinase
Inositol-requiring enzyme 1
Alpha subunit of eukaryotic initiation factor 2
X-box binding protein 1
Indirect immunofluorescence assay
Microtubule-associated protein 2
Glial fibrillary acidic protein
Activating transcription factor 4
Multiplicity of infection.
We especially thank M. Katata for excellent technical assistance. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24591616), the Ministry of Health, Labour and Welfare of Japan (H23-Jisedai-Ippan-001), and the Grants of National Center for Child Health and Development (22A-9 and 24–17).
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