Primary culture of SHED

Human exfoliated deciduous teeth of children were obtained as discarded biological samples collected from children aged 7- to 8-year-old from Department of Pediatric Dentistry of China Medical University. Isolation and characterization of SHED was performed as described previously [19]. To exclude the impact of individual genetic background on our findings, SHED cell lines used in this study were obtained from 10 children respectively. Experiments were conducted from multiple cell lines. Only 4 to 8 generations of SHEDs can be used for experiments and differentiation induction.

Cell preparation

HELFs were cultured in Dulbecco’s minimal essential medium (DMEM, BI, Israel) containing 10% fetal bovine serum (Equitech, USA) and 100U/mL penicillin-streptomycin (BI, Israel) solution. SHEDs were cultured in α-MEM (Gibco, USA) containing 15% FBS (Equitech, USA) and 100U/ml penicillin-streptomycin (BI, Israel) solution.

Trypsin-EDTA (0.25% trypsin, 0.02% EDTA; Gbico, USA) was used for digestion during cell passage. Both HELF and SHEDs were maintained at 37 °C with 5% CO2 and passaged every 3–4 days at 90% confluence.

Virus preparation

Human cytomegalovirus clinical low-passage strain Han and laboratory strain Towne expressing a GFP replorter under the SV40 promoter were derived from BAC DNA as previously described [24]. Towne is a present of Professor Yongjun Yu (Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania, USA). Viral stocks were generated following viral propagation in HELFs by standard ultracentrifugation procedures described previously [25]. The purified virus particles were stored at -80 °C until use.

Differentiation of SHEDs into neural cells

SHEDs were induced to differentiate into neural cells as previously described [18,19,20, 26,27,28]. Briefly, SHEDs were seeded (5000 cells/cm2) on 0.1% Gelatin (STEMCELL, Canada)-coated circular glass cover slips (20 mm in diameter; nest, China) in plates. The attached cells were induced differentiation (1mL/well) in Neurobasal A medium (Gibco, USA) containing 1×B27 (Invitrogen, USA) supplement, 40ng/mL human recombinant basic fibroblast growth factor (bFGF, STEMCELL, Canada), 20ng/mL human recombinant epidermal growth factor (EGF, STEMCELL, Canada) and 100U/mL penicillin-streptomycin. Fresh medium was changed every 24 h. Cells were allowed to differentiate for 7 days or extended to 14 days at 37 °C in an atmosphere of 5% CO2 and 95% O2 prior to fixation and the performance of IFA or other experiments.

Detection of HCMV mRNAs and proteins

To detect viral molecules including HCMV mRNAs and proteins, we infected SHEDs or HELFs with higher multiplicities of infection (MOI) to make the detection of viral molecules better. SHEDs or HELFs were infected by HCMV at MOI of 1, and harvested at 24, 48 and 72 h post infection (hpi). Total RNA was extracted and reverse transcribed using RNeasy Mini Kit (QIAGEN, Germany) and QuantiNova Reverse Transcription Kit (QIAGEN, Germany) according to the supplier’s recommendations. Quantitative PCR was performed for transcripts of HCMV UL123, UL32, UL55, UL44, UL83, UL99 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using QuantiNova SYBR Green Kit (GIAGEN, Germany). Information of primers used in this study was listed in Table S1. Three independent experimental replicates were performed, and the results are presented as means and standard deviation.

HCMV-infected SHEDs were harvested at 24, 48 and 72 hpi by being rinsed PBS, trypsinized, and pelleted. Total cellular protein was extracted by adding an equal volume of M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher, USA) containing protease and phosphatase inhibitor cocktail (ThermoFisher, USA) to cell pellets. HCMV proteins were detected using western blotting with following primary antibodies (Abs): IE2/3 (anti-rabbit, abcam, ab37601), HCMV-gB (anti-mouse, Santacruz, sc-69,742), CMV-pp52 (anti-mouse, Santacruz, sc-69,744), CMV-pp28 (anti-mouse, Santacruz, sc-56,975) and GAPDH (anti-mouse, proteintech, 60004-1-lg). Blots were probed with secondary Abs conjugated with horseradish peroxidase (HRP) and visualized with enhanced chemiluminescence reagents (Millipore, USA). Each experiment was performed in triplicates with representative images shown.

Quantification of HCMV DNA copies in supernatants

SHEDs were infected with HCMV at MOI of 1. Supernatants were collected at 24, 48, 72 and 96 hpi. The DNA copy numbers of HCMV genome in the supernatants were measured using CMV DNA quantification Kit (LifeRiver, China) on QuantStudio Q5 instrument (ThermoFisher, USA). Three independent experimental replicates were set at each time point and the results are presented as means and standard deviation.

Immunofluorescence assay (IFA)

Cells with different treatments including differentiated SHEDs with HCMV infection were first fixed with buffer 4% paraformaldehyde for 30 min at room temperature and blocked with blocking solution (1×PBS containing 0.1% Triton X-100, 0.3 M glycine and 5% BSA) for 1 h. To avoid unwanted morphological changes caused by the overwhelming release of inflammatory cytokines, we infected differentiated SHEDs with a lower MOI of 0.5. Fc receptor blocking solution (absin, abs9476) was added to reduce nonspecific binding prior to incubation with primary antibodies for overnight at 4 °C, respectively. The samples were subsequently washed and incubated with secondary antibodies. Cells were counterstained with DAPI before mounting.

The following antibodies were used for IFA: Nestin (anti-mouse, cell signaling technology, 33475s), β3-tubulin (anti-mouse, cell signaling technology, 5568), MAP2 (anti-mouse, cell signaling technology, 4542), NeuN (anti-mouse, abcam, ab177487), SREBP1 (anti-rabbit, proteintech 14088-1-AP), SREBP2 (anti-rabbit, abcam, ab30682), SCAP (anti-mouse, abcam, ab190103), β-actin (anti-rabbit, abcam, ab179467), Alexa Fluor® 594-labeled Goat Anti-Rabbit IgG (H + L) (ZSGB-BIO, ZF-0516) and Alexa Fluor® 594-labeled Goat Anti-mouse IgG (H + L) (ZSGB-BIO, ZF-0513).

A Carl Zeiss LSM880 confocal microscope with NIS Elements was used for image acquisition and analysis. All of the experiments were performed in triplicates, and representative images were shown. The magnification for all images presented is 400×. Relative fluorescence values of cells in the field of vision were measured and the mean fluorescence value were calculated by Image J software. Mean fluorescence value = Total fluorescence value of the sight/Area of the sight.

Western blots analysis

Cells with different treatments were harvested at indicated time points. An equal volume of M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher, USA) containing protease and phosphatase inhibitor cocktail (ThermoFisher, USA) was added into the cell pellets. Nuclear extracts were prepared using NE-PER™ (ThermoFisher, USA) and cytoplasmic extraction reagents (ThermoFisher, USA) according to the manufacturer’s directions. Protein concentration was measured using BCA protein assay kit (Takara, Japan), equivalent amounts of denatured cell lysate were electrophoresed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, USA). And then the blots were probed with each of the following primary antibodies (Abs): nestin (anti-mouse, cell signaling technology, 33475s), β3-tubulin (anti-mouse, cell signaling technology, 5568), MAP2 (anti-mouse, cell signaling technology, 4542), NeuN (anti-mouse, abcam ab177487), IE2/3 (anti-rabbit, abcam, ab37601), SREBP2 (anti-rabbit, abcam 30,682), HMGCR (anti-rabbit, affinity DF6518), β-actin (anti-mouse, proteintech, 66009-1-lg) and Histone-H3 (anti-rabbit, proteintech, 17168-1-AP). After extensive washing, blots were probed with secondary Abs conjugated with horseradish peroxidase (HRP) to detect bound primary Abs. Proteins were visualized with enhanced chemiluminescence reagents, Immobilon Western HRP Substrate (Millipore, USA). All Western blot experiments were performed in triplicates, with representative images shown.

Transcriptomic sequencing

Cell samples including SHEDs, differentiated SHEDs (SHEDi) and differentiated SHEDs (hSHEDi) with HCMV infection (MOI = 1) were harvested and lysed in TRIzol Reagent (Thermo Fisher, USA) for high throughput RNA-sequencing. After RNA quantification and qualification was assessed, a total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Prepared libraries were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.

Index of the reference genome was built and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Differential expression analysis of two groups (three biological replicates per condition) was performed using the DESeq2 R package (1.16.1). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with Fold Change ≥ 2 and an adjusted P-value < 0.05 found were assigned as differentially expressed.

Differentially expressed genes were functionally categorized and analyzed using Ingenuity Pathway Analysis software (QIAGEN Ingenuity System) and Gene Set Enrichment Analysis (GSEA, http://www.broadlinstitute.org/gsea).

Chemical treatment

U18666A (MCE, HY-107433), a cholesterol synthesis inhibitor [29,30,31], was added to cells at a final concentration of 2 µg/ml at the initiation of differentiation. Fatostatin (MCE, HY-14452), a inhibitor of SREBP activation [32,33,34,35,36], was added to cells at a final concentration of 1µM at the initiation of differentiation. Cholesterol (MCE, HY-N0322) was added into the medium at a final concentration of 10 µg/ml at the initiation of differentiation and supplied to set concentration with the change of medium.

Cholesterol assay

Cells with different treatment were harvested and lysed in M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher, USA) containing protease and phosphatase inhibitor cocktail (ThermoFisher, USA). The samples were diluted with component E (applied) according to the appropriate concentration, and then tested for cholesterol assay including cellular total cholesterol, free cholesterol and cholesterol easter using Amplex® Red Cholesterol Assay Kit (ThermoFisher, USA) according to the manual instruction as previously described [37]. Each assessment was performed in triplicate, and the differences between samples were balanced by protein concentration.

Brdu staining

SHEDs (5 × 103/well) were seeded on glass coverlips and cultured for 2–3 days. The cultures were incubated with BrdU solution (1:100) (Invitrogen) for 20 h, and stained with a BrdU staining kit (Invitrogen) according to the manufacturer’s instructions. The samples were then stained with hematoxylin. BrdU positive and total cell numbers were counted in 10 images per subject. The number of BrdU-positive cells was indicated as a percentage of the total cell number. The BrdU assay was repeated on three independent samples for each experimental group.

Quantitative RT-PCR

Total RNA was extracted by using RNeasy Mini Kit (QIAGEN, Germany) with on column DNase treatment. cDNA was generated from 1 µg total RNA using QuantiNova Reverse Transcription Kit (QIAGEN, Germany) according to the supplier’s recommendations. Quantitative PCR (qPCR) was performed on QuantStudio Q5 instrument (ThermoFisher, USA). Each 20 µl qPCR mixture contained 100ng reverse transcription product, 10 µl 2×QuantiNova SYBR Green PCR Master Mix, 2 µl QN ROX Reference Dye (QIAGEN, Germany), and 0.7µM forward (F) and reverse (R) primers. Primers used in this study were listed in Table S1. Amplification was performed by denaturation at 95 °C for 2 min, followed by 40 two-step cycles of 95 °C for 15s and 60 °C for 30s. Each reaction was performed in triplicates, and the results for the target gene mRNA were normalized to GAPDH using the 2ΔΔCT method. The results are presented as means and standard deviation.

Confocal microscopy

For the co-localization of SREBP and CMV UL44, SHEDs were seeded in cover glass-bottomed 24-well plates and fixed with buffer 4% paraformaldehyde for 30 min at room temperature and blocked with blocking solution (1×PBS containing 0.1% Triton X-100, 0.3 M glycine and 5% BSA) for 1 h. Primary antibodies (SREBP1, Proteintech 14088-1-AP; UL44, Santa Cruz, sc-56,971) were incubated with slides at 4 °C overnight. The samples were then washed and incubated with secondary antibodies.

Images were acquired using a Carl Zeiss LSM880 confocal microscope with NIS Elements. Image J were used for image analysis. All experiments were performed at least in triplicates, and representative images are shown. The magnification for all images presented is 400×.

Statistical analysis

Statistical analyses were performed using Excel and GraphPad Prism 5.0. Data were expressed as mean ± SD. Statistically significant differences were evaluated using unpaired 2-tailed Student’s t test. In all cases, P-value of ≤ 0.05 was considered statistically significant.