Materials
Two kinds of GQDs (i.e. OH-GQDs and NH2-GQDs) were purchased from Xianfeng Nanotechnology Co., Ltd. (Nanjing, China), and their suspensions in water (1 mg/mL and 20 mg/mL, respectively) were used as the stock solutions, and stored in the dark at 4 °C. The working solution was freshly prepared under the sterile condition by diluting with the cell culture medium.
Characterization of GQDs
The morphology of the test GQDs was visualized, using a high-resolution transmission electron microscopy (TEM, JEM-F20, Japan) at the acceleration voltage of 200 kV. Their zeta potentials and hydrodynamic sizes in N2B27 medium at the concentration of 50 µg/mL were analyzed by a Malvern Zetasizer Nano ZS (Malvern, UK) to accord with the cell experiments in the following studies. The fluorescence spectrum of these two GQDs were scanned using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) at the excitation wavelength in the range of 340–410 nm.
Cell culture
The J1 mESCs (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were seeded on 0.1% gelatin (Millipore, USA) pre-coated 96-well, 12-well or 6-well plates at the densities of 1×104, 1×105, and 2×105 cells per well, respectively, and cultured in KSR medium at 37 ℃ under 5% CO2 for 12 h to obtain cell attachment on the plates. Then, the KSR medium was replaced with N2B27 medium for the proliferation and colony formation of the mESCs [43]. The experiments based on the mESCs were performed by adding different concentrations of GQDs in N2B27 medium, and the duration lasted for 24 h or 48 h. The naïve ESCs were also tested throughout the study as the undifferentiated control for evaluating the neural committed differentiation process.
Cell viability
The mESCs cultured in 96-well plates were exposed to a series of concentrations of OH-GQDs or NH2-GQDs (0, 1, 10, 50 µg/mL) for 24 h and 48 h, respectively. When the exposure was terminated, the cells were incubated with the fresh N2B27 medium containing 10 µM alamarBlue (Thermo Fisher Scientific, USA) for 2 h at 37°C in the dark, and the absorbance at the wavelength of 490 nm was recorded using a microplate reader (VARIOSKAN FLASH, Thermo Fisher Scientific, USA). The non-cytotoxic levels of GQDs based on the results of this assay were selected for the following mESC experiments.
EdU incorporation assay
The cellular proliferation of the mESCs was evaluated by EdU incorporation method. Briefly, the mESCs were exposed to different concentrations of GQDs (0, 1, 10 and 50 µg/mL) for 48 h, and the probe of EdU (2.5 µM) was added to the exposure systems and incubated for 2 h to label the S-phase cells with DNA replication. Then, the cells were harvested, fixed, permeabilized, and stained in accordance with the manufacturer’s instructions (RiboBio, China). The quantitative analysis of apollo 567-labeled EdU-positive cells in different exposure groups were examined using a flow cytometry (NovoCyte, Agilent Technologies, USA).
Alkaline phosphatase (AP) activity
According to the previously-reported protocol [43], the mESCs were submitted to 48-h GQD exposure (0, 1, 10 and 50 µg/mL) in 12-well plates, and then fixed with citrate-acetone-formaldehyde buffer, incubated with alkaline-dye mixture and finally counterstained with hematoxylin solution using the AP kit (Sigma, USA). The morphology of the cell colonies from different exposure groups was observed under the inverted microscope (Olympus IX73, Japan), and the representative images were taken from different visual fields.
EB formation assay
In vitro differentiation of the mESCs into EBs was performed according to the protocol reported previously [44]. Briefly, the mESCs were passaged using 0.05% TrypLE express enzyme, seeded onto ultra-low attachment 6-well plates (Corning, USA) at the density of 4⋅105 cells per well, and cultured in 2 mL of differentiation medium containing 1 and 10 µg/mL OH-GQDs or NH2-GQDs. The controls without GQD treatment were also designed, and the differentiation medium was replaced every other day during EB formation. The EB samples were collected on differentiation day 4 and 9 for the subsequent characterization of three germ layers using the quantitively polymerase chain reaction (qPCR) assay. The mESCs were also tested as the undifferentiated control.
Neural differentiation of the mESCs
Briefly, the mESCs (8×104 cells per well) were seeded onto 0.1% gelatin-coated 6-well plates, and cultured in N2B27 medium consisted of 50% DMEM/F12 and 50% Neurobasal™ medium (Gibco, USA), supplemented with 0.1% bovine serum albumin fraction V (Beyotime, China), 1× N2 supplement, 1× B27 supplement (Gibco, USA), 0.1 mM β-mercaptoethanol and 1% GlutaMax). The neural differentiation media was changed every other day, and the exposure experiments were performed by culturing the mESCs in the neural differentiation media, containing different levels of OH-GQDs or NH2-GQDs. The cell samples exposed to 10 µg/mL GQDs were collected on differentiation day 2, 6 or 12 for nerve formation and BMP signaling evaluation, using qPCR. The cells harvested from GQD treatments at different exposure levels (0, 1, 10 µg/mL) on neural differentiation day 12 were submitted to immunofluorescence staining, chromatin immunoprecipitation (ChIP) analysis or western blotting. The mESCs were concomitantly tested for the comparison with the neural differentiated cells.
Immunofluorescence staining
The neural cells from the committed differentiation of the mESCs on day 12 were fixed, and incubated overnight with MAP2 (1:50, Cell Signaling Technology, USA) or β3 Tubulin antibodies (1:50, Santa Cruz Biotechnology, USA). Then the cells were immunoblotted with Alexa fluorescently-labeled IgG antibody (1:1000, Proteintech, China) for 1 h, and finally stained with DAPI. The representative images were captured under different visual fields using a fluorescence microscope (Olympus IX83, Japan).
The qPCR analysis
Total RNA samples were extracted from different treatments described above using the TRIzol Reagent (Gibco, USA), and submitted to the synthesis of first-strand complementary DNA (cDNA) with a reverse transcription kit (BioRad, USA). The quantification of the target gene expressions was carried out using SYBR Green qPCR Master Mix (BioRad, USA) on Roche 480 Real-Time PCR system (Roche, USA). The relative transcriptional levels of the biomarkers for EB formation (Mesp1, Brachyury T, Gata6, Sox17, Fgf5 and Krt14), neural differentiation (Sox1, Sox3, Pax6, Map2, NeuroN and Dcx) and BMP signaling (Smad6, Smad9 and Id3) were normalized to that of GAPDH, using the method of 2−△△CT, and the primer sequences for these test genes were listed in Additional file 1: Table S1.
Western blotting assay
The cell samples after 12-day neural differentiation, together with the undifferentiated mESCs, were lysed with RIPA solution (Solarbio, China). The extracted proteins were subsequently separated by the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred to a PVDF membrane. The target proteins were investigated by the overnight incubation of the membrane with the corresponding primary antibodies, including anti-Map2, anti-Smad1, anti-p-Smad1/5/9, anti-H3K27me3 (1:1000, Cell Signaling Technology, USA) or anti-GAPDH (1:2500, Abcam, USA). The horseradish peroxidases (HRP)-labeled secondary antibody (1:2500, ZSGB-BIO, China) were subsequently used. The quantitative analysis of the target protein expressions was performed by adjusting with the corresponding GAPDH levels.
Chromatin immunoprecipitation (ChIP) analysis
The ChIP analysis was performed to investigate the enrichment of H3K27me3 at the Smad6 promoter, according to the manufacturer’s instructions (Cell Signal Technology, USA). The pair primer sequences for the ChIP were listed in Additional file 1: Table S1 for the binding site. Briefly, the cells from different treatments were fixed with formaldehyde to cross-link DNA and proteins. After the wash with cold PBS twice, the cells were lysed with SDS buffer, and the genomic DNA was sonicated into fragments. After the incubation with the antibodies against IgG or H3K27me3, DNA was purified and amplified by the specific primers (Additional file 1: Table S1) using qPCR assay.
Statistical analysis
All the experiments were independently performed for 3 times or more. The data from different treatments were presented as the mean ± SE, and analyzed with one-way ANOVA followed by LSD's post hoc analysis. The significant differences were considered in all tests, when p value was less than 0.05, 0.01, or 0.001.