Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Further, to consider the potential influence of off-target gene modifications (Yee, 2016), we detected the most probable ten off-target modification sites which were designed at the CRISPR website (http://crispr.mit.edu/↗) (Table S3) and found that there was no off-target gene modification (Fig. S2), which excluded the potential influence of off-target gene modifications.

Mouse 129 ESCs were obtained from ATCC (American Type Culture Collection, Manassas, VA). The wild type and Clock knockout mESCs were cultured in the maintenance medium of Dulbecco’s Modified Eagle’s Media (DMEM, Gibco, USA) supplemented with 3.7 g/L sodium bicarbonate, 1% penicillin and streptomycin, 1.7 mmol/L L-glutamine, non-essential amino acid, 55 mmol/L beta mercaptoethanol, 5 ng/mL mouse leukemia inhibitory factor (LIF), 3 μmol/L EsK3β inhibitor, 1 μmol/L MEK inhibitor and 10% FBS. The mESC maintenance medium was used for spontaneous differentiation without supplementing LIF, the EsK3β inhibitor or the MEK inhibitor.

To construct the Clock knockout mESC line, we utilized the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CAS9) (CRISPR/CAS9) genomic editing tool. The CRISPR/CAS9 system is an efficient tool for genome engineering. It induces double-strand breaks (DSBs) and repair using the non-homologous end-joining (NHEJ) mechanism at any specific site. Briefly, one pair of independent oligo primers targeting exon 2 of the Clock gene was subcloned into the pX330 vector (Mizuno et al., 2014) (Addgene) to obtain the pX330-Clock plasmid. The primer sequences generating the guide RNA (gRNA) targeting the Clock gene were TCCATCTTTCTCGCGTT, then the complementary sequences were AGGTAGAAAGAGCGCAA. As the transcriptional promoter of PX330 plasmid was U6, we replaced base T to base G for the first base was G to be effective transcription. With enzyme sites, the insertion sequences were as follows: oligo1: 5′-CACCGCCATCTTTCTCGCGTTACC-3′, oligo2: 5′-AAACGGTAACGCGAGAAAGAT GGC-3′. The pX330-Clock plasmid was transfected into single mESCs by electroporation. The correct knockout of Clock gene expression was confirmed by Western blot analysis and gene sequencing.

To consider the potential influence of off-target gene modifications (Yee, 2016), we detected the most probable ten off-target modification sites which were designed at the CRISPR website (http://crispr.mit.edu/↗), whose off-target cleavage site sequences and gene numbers were as follows in Table S3. Specific PCR primers were designed according to the target gene sequences of mouse (Table S3).

The mRNA expression levels of GAPDH, Oct4, Sox2, Klf4, Nanog, Zfp296, Eras, Dax1, Esg1, C-Myc, PCNA, CDK1, CDK2, CyclinD1, P27, Gata4, Sox17, Foxa2, Lamb1, BMP4, T, Eomes, Actc1, Nestin, Sox1, Neurod1, Otx1, Bcl-2, Bax, cleaved caspase-3, and caspase-9 were quantified by RT-PCR. Specific PCR primers were designed according to the target gene sequences of mouse (Supplemental Table. 1). Cells were washed by ice-cold Phosphate buffer solution (PBS, NaCl 137 mmol/L,KCl 2 mmol/L, Na₂HPO₄ 10 mmol/L, KH₂PO₄ 10 mmol/L pH 7.4). Total RNA was extracted by using Trizol reagent (Invitrogen, USA). Qualities of extracted RNA qualities were measured by OD₂₆₀/OD₂₈₀ ratios which ranged from 1.9 to 2.1. The cDNA was obtained through reverse transcription kit according to manufacturer’s instructions (TOYOBO, Japan). The amplification mixture comprised 1 μL of RT reaction mix, 10 μL of SYBR® Premix Ex Taq TM (2×) (TaKaRa, China), 0.5 μL of 10 μmol/L each of primers and 8.5 μL ddH₂O. Reactions were performed on a fluorescence temperature cycler (Bio-Rad, Hercules, CA, USA). The PCR conditions for Clock and GAPDH were as follows: one cycle of 3 min at 95°C; 35 cycles of 15 s at 95°C, 30 s at 58°C, 30 s at 72°C. The primer sequences are listed in Supplemental Table 1. The threshold cycle (CT) in RT-PCR was analyzed using the 2^-ΔΔCt method

Total protein was extracted from the treated cells using Radio-Immunoprecipitation Assay (RIPA) lysis buffer (50 mmol/L Tris/HCl pH 7.4, 150 mmol/L NaCl, 1% Nonidet-P40, 0.5% Sodium deoxycholate, 0.1% SDS). The extraction and isolation of nuclear protein were performed according to the instructions in the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Equal amounts (30–50 μg) of protein extracted from cells were boiled at 100°C in 1× SDS loading buffer for 10 min and then were loaded and separated by sodium dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), blocked by 5% nonfat dry milk in Tris-buffered saline containing Tween-20. The membranes were incubated with antibodies against PKM2 (Cell Signaling), CK19 (Abcam), and EpCAM (Abcam). Signals were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit IgG. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences), which was developed and quantified using an imaging system (Tanon, China).

The sections were washed with cold 1 × PBS three times, treated with 4% paraform- aldehyde for 1 to 2 min, washed with cold 1 × PBS twice, and with 1 × Tris buffer solution tween (TBST, 50 mmol/L Tris, 150 mmol/L NaCl, 1% Tween-20) once, followed by staining with Alkaline Phosphatase Kit (Millipore, USA) according to the manufacturer’s instructions. Images were taken by a Leica DMi8 microscope (Leica, Germany) and analyzed using Image Pro Plus 6.0 software (Media Cybernetics, Rockville, MD).

To study the pluripotency of wild type mESCs and Clock knockout mESCs in vivo, we performed teratoma formation assay which could conduct as a tool for monitoring pluripotency in stem cell research (Nelakanti et al., 2015). We injected 1 × 10⁷ wild type mESCs and Clock knockout mESCs into the oxters of immunodeficiency mice respectively. 1–2 cm teratoma formed after four weeks. Six weeks later, we detected the differential capacity of wild type mESCs and Clock knockout mESCs by hematoxylin-eosin staining (HE staining). We found that both wild type mESCs and Clock knockout mESCs could differentiate to endoderm, mesoderm, and ectoderm layers cells, which indicated that they have similar potential of multilineage differentiation in vivo.

The sections were washed with cold 1 × PBS three times, and treated with 4% paraformaldehyde for 15 min. They were again washed with cold 1 × PBS three times, and treated with 5% Triton X-100 in Tris buffer solution (TBS, 50 mmol/L Tris/HCl pH 7.4, 150 mmol/L NaCl) for 20 min at room temperature (RT) (membrane proteins do not need this step). After another wash with cold 1 × PBS three times for 5 min, they were treated with normal goat serum for 30 min at RT, followed by the addition of primary antibodies (1:200) and incubation in a wet box for 2 h at RT or 4 °C overnight. Fluorescent secondary antibodies (1:350) were added and the cells were incubated for 30 min or 1 h at RT, washed three times for 5 min each with PBST (1% Tween in PBS buffer). It was followed by staining with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (1:1000) for 5 min at RT, and washed three times for 5 min each with PBS. After treatment with a fluorescence quenching agent, the specimens were sealed and photographed under fluorescence and laser confocal microscope. The images were analyzed using Image Pro Plus 6.0 software (Media Cybernetics, Rockville, MD).

Cell viability and number was detected using a cell counting chamber. Wild type mESCs and Clock knockout mESCs were transferred to 12-well and 6-well plates (Corning Inc., Corning, New York, USA) at 1 × 10⁴ cells/well and cultured at 6 different time points (24, 48, 72, 96, 120 and 144 h). At the indicated time, cells were digested with 0.05% trypsin, and 20 μL of the resuspended cells were collected in the cell counting chamber (Nexcelom) and counted using the Cellometer Mini software.

Briefly, we induced the biological rhythms of wild type mESCs and Clock knockout mESCs after spontaneous differentiation with horse serum shock as previously described (Balsalobre et al., 1998; Xiang et al., 2012). The wild type mESCs and Clock knockout mESCs were cultured in the mESC maintenance medium and differentiated spontaneously for 15 days in the differentiation medium. The medium was removed and cells were washed with 1 × PBS, followed by addition of the differentiation medium without FBS for 24 h. The medium was removed and cells were washed with 1 × PBS. A new differentiation medium containing 50% horse serum was added to the cells. After incubation for 2 h, the medium was removed and washed with 1 × PBS, and replaced with the original differentiation medium.

The FITC Annexin V and Propidium Iodide (PI) Apoptosis Detection Kit II (Pharmingen, USA) were used to analyze apoptosis and cell death rate using flow cytometry. The wild type mESCs and Clock knockout mESCs were collected and washed twice with cold PBS. The resuspended cells in 100 μL of 1 × binding buffer were transferred to a 1.5 mL culture tube, and 5 μL of FITC Annexin V and PI were added to each tube. After gentle vortexing, the cells were incubated for 15 min at room temperature in the dark. Finally, 400 μL of the 1 × binding buffer was added to each tube, and analyzed by flow cytometry in 1 h.

All the data were expressed as means ± SD. The statistical significance of differences between the experimental groups was determined using one-way ANOVA by Student’s t-test (SPSS 11.0, SPSS Inc., Cary, NC). Probability values (p) of less than 0.05 were considered statistically significant.

Below is the link to the electronic supplementary material. Supplementary material 1 (PDF 392 kb) Supplementary material 2 (PDF 220 kb)