Cell culture

E14 mESCs were purchased from the Chinese Academy of Sciences Stem Cell Bank. They were cultured at 37 °C in 5% CO₂ in an incubator. The culture medium was Knockout DMEM (Gibco, 10829-018), added with 15% fetal bovine serum (FBS, Gibco, 10099-141), 1× MEM nonessential amino acids (Gibco, 11140-050), 1× GlutaMAX (Gibco, 35050-061), 50 μM 2-Mercaptoethanol (Sigma, M3148), 1000 IU/mL LIF (Millipore, ESG1107), 1 μM PD0325901 (Selleck, S1036) and 3 μM CHIR99021 (Selleck, S2924) [25]. mESCs were maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs) for passage or in 0.1% gelatin-coated dishes for assays. The medium was changed every day, and the cells were passaged every 3 days.

Haspin knockout in mESCs

To knockout Haspin, we designed two guide RNAs (gRNAs) targeting Haspin coding sequences. The gRNAs were annealed and then cloned into the BbsI-digested vector pX459. These two Haspin gRNA plasmids were co-transfected into WT E14 cells using Lipofectamine 2000 (Lip2000, Invitrogen, 11668019). 24 h after transfection, the medium was changed to ES medium supplemented with 1.5 μg/mL puromycin for 3 days, and the surviving single clones were picked and transferred to 96-well plates. To obtain Haspin-KO cells, we used immunostaining of H3T3ph to select H3T3ph negative single clones. Then, the genomic DNA fragments of the Haspin-KO cells were amplified by PCR and sequenced to confirm that the Haspin gene was disrupted.

Induction of asymmetric stem cell division

When assaying asymmetric stem cell division ability, E14 mESCs were switched to N2B27 medium and incubated for 16 h on gelatin-coated glass-bottom cell culture dishes (Nest, 801001) after passage. N2B27 medium includes DMEM/F12 (Gibco, 11320-033), Neurobasal (Gibco, 21103-049) (1:1), 50 μM 2-mercaptoethanol, 0.5% N2 (Gibco, 17502-048), 1% B27 (Gibco, 17504-044), 0.033% BSA 7.5% solution (Thermo Fisher, 15260037) and 1× GlutaMAX (Gibco, 35050-061) [24]. After 16 h, immunofluorescence staining experiments were immediately performed on the cells, and then the cells were prepared for confocal imaging. Nanog, one of the core pluripotency markers, has always been used to determine ACD ratio [24,25,26]. When the signal ratio of Nanog between the two daughter cells is greater than 1.2, it is considered as ACD [53].

Plasmid construction and overexpression

The Chek2-eGFP and Chek2-RFP vectors were obtained from Dr. Zhiyong Mao. We amplified the coding sequences of Haspin and Wnt5a from cDNA of mESCs and then subcloned them into the Chek2-eGFP vector to displace the Chek2 coding sequence. For overexpression, Haspin-eGFP or Wnt5a-RFP vector was transfected into E14 mESCs using Lip2000. After 3 days of transfection, GFP-positive cells were purified with flow cytometry using green fluorescence. The efficiency of overexpression was measured by qRT‒PCR and western blotting.

siRNAs

E14 cells were transfected with siRNA targeting Haspin or Wnt5a for 48 h using Lip2000 in accordance with the manufacturer’s instructions. The Haspin and Wnt5a siRNAs were synthesized by Ribobio (Guangzhou, China), and their target DNA sequences are shown in Supplemental Table 1.

Haspin kinase inhibitor CHR-6494

The Haspin kinase inhibitor CHR-6494 (MCE, HY-15217) was dissolved in DMSO to generate a 10 mM stock solution and then stored at −20 °C [27]. After cell passage and adherence, the medium was changed to 0.5 μM or 1 μM CHR-6494 added ES medium or N2B27 medium, and 1 μM DMSO was used as a control. The medium was changed every day.

Cell synchronization

To measure the level of H3T3ph, we synchronized the mESCs. Cells were synchronized to metaphase through treatment with 1 μg/mL colchicine (Sigma, C3915) for 8 h, and then, the cells were collected for western blotting or immunofluorescence staining.

Alkaline phosphatase staining

mESC alkaline phosphatase (AP) staining was performed according to the manufacturers’ instructions using the Alkaline Phosphatase Detection Kit (Millipore, SCR004). First, the cells were fixed for 1 min at room temperature with 4% paraformaldehyde in PBS and then washed for 5 min with 1× PBST. Next, Fast Red Violet solution and Napthol AS-BI phosphate solution were applied to the mESCs for 10 min in the dark. Stained cells were washed with 1× PBS and examined under an inverted microscope.

Growth curves

Approximately 20,000 cells were seeded into each well of 24-well plates (Thermo, 142475). Then, growth curves were generated at 24, 48, 72, 96, and 120 h, and three wells of each line were counted to determine the cell number. Cell number was measured via Countess II (Thermo) according to the manufacturer’s protocol. The remaining cells were washed with 1× PBS, and the medium was replaced daily with fresh medium.

Cell cycle analysis

E14 mESCs were plated in a six-well cell culture plate (Thermo, 140675). Three days later, the cells were trypsinized and fixed in cold 70% ethanol overnight at 4 °C. The cells were resuspended and incubated with RNaseA solution at 37 °C for 30 min, followed by incubation with 50 μg/mL of propylidine iodide (PI) solution for another 30 min at room temperature. Then, BD FACS Aria ll was used to assess the frequency of cells in the G1 phase, S phase, and G2/M phase.

Embryoid body generation

The hanging drop method was used to generate embryoid bodies (EBs). E14 cells were dissociated and resuspended in EB medium at a concentration of 1–1.5 × 10⁵ cells/mL. EB media containing Knockout DMEM, 15% FBS, 1× MEM nonessential amino acids, 1× Sodium Pyruvate, and 0.1 mM 2-Mercaptoethanol [54]. Then, 20 μL hanging drops were cultured for three days in the lid of a 10 cm dish. To prevent the hanging drop from drying out, 1× PBS was placed at the bottom of the lid. After three days, EBs were collected, transferred into ultralow adhesion plates (Corning, 3474) and cultured in EB media for another 3 days. EB samples were collected and then subjected to qRT‒PCR to analyze the gene expression levels.

Teratoma formation

Mice were housed and treated according to the Animal Research Institute Committee guidelines of Tongji University, Shanghai, China. For teratoma formation, E14 cells were trypsinized to produce single cells. For each cell type, a total of 1 × 10⁶ cells were resuspended in 100 μL EB medium and subcutaneously injected into the two flanks of immunodeficient NOD-SCID 8-week-old male mice (n = 4 tumors/group). Four weeks after inoculation, the teratomas were harvested and weighed. Then, these teratomas were collected in 4% PFA, embedded in paraffin, and sectioned in a microtome. Sections of each teratoma were stained with hematoxylin/eosin (H&E) and further analyzed.

Quantitative real-time PCR (qRT‒PCR)

Total RNA was extracted from WT and Haspin-KO mESCs, EBs and teratomas using TRIzol (Invitrogen, 10296010) according to the protocol. Total RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo). Then, reverse transcription into cDNA was performed by the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047). qRT‒PCR was performed using TB Green Fast qPCR Mix (Takara, RR430) with a reaction volume of 20 μL and the Bio-Rad CFX Connect system (Bio-Rad). The relative mRNA expression levels were normalized to GAPDH and analyzed using the 2-delta-delta-Ct method to calculate the relative fold change. Primer sequences are listed in Supplementary Table 1.

Immunofluorescence staining

For immunofluorescence staining, cells grown on glass-bottom cell culture dishes were fixed with 4% paraformaldehyde at room temperature for 30 min. The samples were permeabilized with PBST (0.2% Triton X-100) at room temperature for 30 min. Then, the cells were blocked with 5% BSA at room temperature for 1 h. Then, the samples were incubated overnight with primary antibodies at 4 °C. Furthermore, the samples were washed three times with PBST and incubated at room temperature for 1 h with secondary antibodies and Hoechst 33342 (Invitrogen, H3570). Then, the samples were washed in PBST three times at room temperature and stored in the dark. The images were captured using a Zeiss-LSM880 inverted microscope. Data were processed using Zeiss software and ImageJ software.

Western blotting

For protein extraction, mESCs were lysed using RIPA buffer (Beyotime, P0013B) supplemented with protease inhibitors (Beyotime, P1005) and phosphatase inhibitors (Beyotime, P1081), and then, the lysates were centrifuged at 12,000 rcf and 4 °C for 20 min. The protein concentration of the supernatant was determined using the BCA Protein Assay (Thermo, 23227) and then normalized. Equal amounts of protein samples were separated using 15% SDS‒PAGE gels and then transferred to 0.2 μm PVDF membranes (Millipore, ISEQ00010). Membranes were blocked with TBST supplemented with 5% BSA at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. Next, the membranes were washed three times using TBST followed by HRP-conjugated secondary antibodies for 1 h at room temperature. The membranes were visualized with the chemiluminescence reagent ECL (Thermo, 34577).

RNA-seq and data analysis

RNA was extracted from Haspin-KO and control mESCs using TRIzol. Then, 1 μg RNA was prepared to generate a sequencing library. Libraries were sequenced on the IIIumina NovaSeq 6000 (Berry genomics) according to the protocol. For data analysis, the trimmed reads (using Cutadapt, v1.9.1) were mapped to the mouse genome (mm10) using Hisat2 (v2.0.1). Differentially expressed gene (DEG) analysis was performed using DESeq2. The significant DEGs were identified with P values < 0.05 and fold change >2, and then GOSeq (v1.34.1) was used to identify Gene Ontology (GO) terms.

Dual-luciferase reporter assay

We amplified fragments of the Wnt5a promoter (~2 kb upstream of the TSS) and Pax2 CDS, and then, we cloned Wnt5a into the pGL3 luciferase reporter vector, and Pax2 CDS into the CAG-eGFP overexpression vector. mESCs were seeded in 24-well plates at a density of 2 × 10⁵ cells/mL. The next day, Pax2-eGFP, pRL-SV40, pGL3-Basic, or pGL3-Wnt5a promoter were co-transfected into cells using Lip2000 and cultured for 36 h. On the other hand, pRL-SV40 and pGL3-Wnt5a promoter were co-transfected into WT cells. After 8 h, the transfected cells were administered medium to containing WT, DMSO, 0.5 μM CHR-6494, and 1 μM CHR-6494 and incubated for 36 h. Then, the cells were harvested and lysed to measure the luciferase activity according to the manufacturer’s protocol (Promega, E1910).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed by the Magna ChIP A/G kit (Millipore) according to the protocol. Briefly, mESCs were cross-linked and then sonicated into 200–500 bp fragments, which were next immunoprecipitated with antibodies against Pax2 (Cell Signaling Technology, 9666) or IgG. Then precipitated chromatin DNA was then recovered and analyzed by qRT‒PCR assays.

Antibodies

The following primary antibodies were used for western blotting or immunofluorescence staining: anti-H3T3ph (Abcam, ab130940), anti-H3S10ph (Abcam, ab14955), anti-GAPDH (Proteintech, HRP-60004), anti-caspase-3 (Cell Signaling Technology, 9662S), anti-cleaved caspase-3 (Cell Signaling Technology, 9664S), anti-α-tubulin (Active motif, 39527), anti-Nanog (Abcam, ab80892), anti-Sox2 (Abcam, ab79351), anti-Oct4 (Abcam, ab27985), anti-Klf4 (Abcam, ab129473), anti-Wnt5a (Abcam, ab235966), and anti-Pax2 (Cell Signaling Technology, 9666).

Statistical analysis

All the statistical data are presented as the mean ± SEM. Each test was repeated at least three times, and the results were analyzed by SPSS 20.0. Statistical significance between groups was determined using unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant differences, and P > 0.05 was considered not significant.

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• Source: https://www.nature.com/articles/s41420-023-01604-w