NPCs and human embryonic lung fibroblast cells (HELs) were isolated and cultured as previously described (Pan et al., 2013). Briefly, NPCs were proliferated in fibronectin (14.28 μg/mL, Millipore) coated tissue culture dishes, then digested with accutase (Millipore) and transferred to poly-D-lysine (50 μg/mL, Millipore) coated dishes for further experiments. NPCs growth medium (GM) was Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12) containing amphotericin B (1.5 μg/mL), L-glutamax (2 mmol/L), penicillin-streptomycin (100 U/mL), Gentamicin (50 μg/mL), human basic fibroblast growth factor (bFGF, 20 ng/mL), human epithelial growth factor (EGF, 20 ng/mL) (Gibco) and 10% BIT9500 serum substitute (Stem Cell Technologies). The medium for NPCs culture was half changed with fresh GM every two days, and the old medium was collected as condition medium (CM).
For the culturing of HELs and 293T cells (ATCC #CRL-321), Minimum essential medium (MEM) and Dulbecco modified Eagle medium (DMEM) were utilized, respectively, with the supplementation of 10% fetal bovine serum (Gibco/Life Technology), glutamine (2mmol/L, Gibco/Life Technology), and penicillin-streptomycin (100 U/mL and 100μg/mL). Cells were cultured in appropriate medium at 37 °C in a humidified atmosphere containing 5% CO2.
HCMV Towne strain (ATCC-VR977) were propagated in HEL as described previously (Fu et al., 2015). Briefly, HEL (6 × 106) were infected with the virus at a multi plicity of infection (MOI) of 0.02, and the supernatants were harvested at 7 and 10 days post infection (dpi). Viruses were concentrated by ultracentrifugation at 4 °C (25, 000 × g for 4 h), resuspended in GM containing 1% dimethyl sulfoxide (DMSO), titrated by plaque assay as described previously (Casavant et al., 2006), and stored at –80 °C as aliquots.
For HCMV infection, NPCs were cultured in GM without BIT9500 for 24 h to synchronize the cell status. Then, the synchronized cells were infected by virus at an MOI of 3. 3 h later, the inoculum soup was replaced with culture medium consisting of fresh GM and CM at equal volume. Cells were harvested for further assays at the indicated time points.
Total RNA was isolated from the harvested cells using Trizol Reagent (Takara), quantitated by NanoDrop 2000 Spectrophotometer (Thermo Co., USA), and reverse-transcribed using PrimeScript RT Master Mix (TaKaRa). Quantitative real-time PCR reactions were performed using iTaq Universal SYBR Green Supermix on a CFX Connect Real-Time PCR Detection System (Bio-Rad). Each sample was run in duplicate with GAPDH as the normalizing reference. The primers for qRT-PCR is as following: Hes1, F-ACCCCAGCCAGTGTCAAC, R-GAATGTCCGCCTTCTCCA; GAPDH, F-GAAGGTGAAGGTCGGAGTC, R-GAAGGTGAAGGTC GGAGTC.
Primers Sequence scramble Forward Primer (5′-3′) UUCUCCGAACGUGUCACGUTT Reverse Primer (5′-3′) ACGUGACACGUUCGGAGAATT Hes1-1 Forward Primer (5′-3′) CGGCCAAUUUGCCUUUCUCTT Reverse Primer (5′-3′) GAGAAAGGCAAAUUGGCCGUC Hes1-2 Forward Primer (5′-3′) CGACACCGGAUAAACCAAATT Reverse Primer (5′-3′) UUUGGUUUAUCCGGUGUCGUG Hes1-3 Forward Primer (5′-3′) CACUGAUUUUGGAUGCUCUTT Reverse Primer (5′-3′) AGAGCAUCCAAAAUCAGUGTT Hes1-4 Forward Primer (5′-3′) GCCUAUUAUGGAGAAAAGATT Reverse Primer (5′-3′) UCUUUUCUCCAUAAUAGGCTT
Table 1. Primers for siRNA
Cells were harvested with cell scraper, pelleted by centrifugation, resuspended in ice-cold PBS, snap frozen in liquid nitrogen, and stored at –80 °C. The cells then were lysed in RIPE buffer (50 mmol/L Tris-base, 1.0 mmol/L EDTA, 150 mmol/L NaCl, 0.1% SDS, 1% tritonX-100, 1% sodium deoxycholate and cOmplete Protease In hibitor [Roche]). The protein concentration was measured by the BCA kit (Beyotime) according to the manufacturer’s protocol. Samples with equal amount of protein were separated by SDS-PAGE gel and trans ferred to PVDF membranes (0.25 μm, Millepore). After incubation with the indicated primary and corresponding secondary antibodies, signals were detected using a Chemiluminescence machine, and analyzed by den- sitometry program (Image J). At least three sets of in dependent experiments were performed and repre- sentative results were shown. The primary antibodies used included mouse monoclonal antibodies against -Hes1 (IgG1, Abcom), -GFAP (IgG2b, Fitzgerald In dustries International, Inc.), -Nestin (IgG1; Chemicon), -SOX2 (cell signal, IgG1), -DCX (IgG; Santa Cruz Biotechnology) and β-actin (Santa Cruz Biotechnology). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Bioscience).
To determine the half-life of Hes1 protein (t1/2), NPCs were treated with 0.1mg/mL cycloheximide (CHX, Sigma-Aldrich) (Ajiro and Zheng, 2015) and collected at the indicated time points, Hes1 protein levels were enriched by Immunoprecipitation (IP), and then examined by IB. Hes1 degradation curve was generated by the log2 of the relative Hes1 protein level (Hes1/β-actin) normalized to that of 0 min (y) versus sampling time (x). The half-life of endogenous Hes1 protein were measured and calculated as previously described (Hirata et al., 2002).
siRNAs were synthesized at GenePharma company, and the sequences were listed in Table 1. 293T cells transfected with Hes1 expressing construct (pCDH-Hes1) applied to assess the knocking down efficiency of the siRNAs. Briefly, 4 × 106 293T cells were seeded in 6-well plate one day prior to transfection. 100 pmoL siRNA and pCDH-Hes1 or 5 μL Lipofectamine (Invitrogen) were diluted to a total volume of 50 μL, respectively. After 5 minites incubation, the diluted DNA and Lipofectamine were mixed, and incubated for 20 minutes at room temperature. The mixture was then added to 293T cells and the medium was changed 4 h later. Cells were harvested 48 h later to examine the Hes1 protein amount.
siRNA were introduced to NPCs by Nucleofection, which was performed using Amaxa Mouse NSC Nucleofector Kit (Lonza) following the manufacture's instruction. Briefly, 110 μL reaction medium was prepared by mixing Nucleofector solution (82 μL), supplement (18 μL) and siRNA (scr and si-3, 100 pmol). NPCs were resuspended in the reaction medium, transferred into a certified cuvette, applied to Nucleofector Program A-033, NPCs were then cultivated in uncoated dishes as neurospheres or in poly-D-lysine-coated dishes as monolayers (Li et al., 2015).
NPCs nucleofected with siRNA were cultured on uncoated or poly-D-lysine-coated culture dishes for 48 h. Then the images were obtained using a Nikon Eclipse TS100 inverted microscope equipped with a Nikon CoolPix P6000 camera. Representative images were collected from three independent experiments. The monolayer NPCs on coated surface were digested with accutase for 1 to 2 min, pelleted by centrifugation, resuspended and counted using a cell-count board. The neurospheres were categorized by diameter into three size groups (small, < 50 μm; medium, 50–100 μm; and large, > 100 μm).
All the results are representative of at least three in-dependent experiments. Differences were considered statistically significant at P ≤ 0.05 based on a Student’s t-tests analysis.
Human cytomegalovirus infection dysregulates neural progenitor cell fate by disrupting Hes1 rhythm and down-regulating its expression
- Received Date: 16 February 2017
- Accepted Date: 28 March 2017
- Published Date: 24 April 2017
Abstract: Human cytomegalovirus (HCMV) infection is a leading cause of birth defects,primarily affecting the central nervous system and causing its maldevelopment.As the essential downstream effector of Notch signaling pathway,Hes1,and its dynamic expression,plays an essential role on maintaining neural progenitor/stem cells (NPCs) cell fate and fetal brain development.In the present study,we reported the first observation of Hes1 oscillatory expression in human NPCs,with an approximately 1.5 hour periodicity and a Hes1 protein half-life of about 17(17.6±0.2) minutes.HCMV infection disrupts the Hes1 rhythm and down-regulates its expression.Furthermore,we discovered that depleting Hes1 protein disturbed NPCs cell fate by suppressing NPCs proliferation and neurosphere formation,and driving NPCs abnormal differentiation.These results suggested a novel mechanism linking disruption of Hes1 rhythm and down-regulation of Hes1 expression to neurodevelopmental disorders caused by congenital HCMV infection.