Reagents and media
All the reagents and media used in our experiments were purchased from Life Technologies or Stem Cell, Inc., unless otherwise indicated. MEFs were purchased from the Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences.
Cell culture
Human haESCs were a kind gift from Dr. Jinsong Li of the State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences. The haESCs were cultured as previously reported [15, 25, 34]. MEFs were treated with 10 µg/mL mitomycin C for 3 hours to generate a feeder layer and cultured in 10% FBS (BI) plus 1% penicillin‒streptomycin DMEM. The ES medium before optimization contained DMEM/F12 + 20% KNOR + 0.1 mM NEAA, 100 U/ml penicillin-0.1 mg/ml streptomycin, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, and 10 ng/ml bFGF. Human EPS cells were cultured in N2B27-LCDM medium under 20% O2 and 5% CO2 at 37°C. Then, 50 ml of N2B27 medium was prepared and included the following: 24 mL of DMEM/F12 (Gibco), 24 mL of Neurobasal (Gibco), 0.25 mL of N2 supplement (Gibco), 0.5 mL of B27 supplement (Gibco), 1% GlutaMAX (Sigma‒Aldrich), 1% NEAA (Gibco), 0.1 mM β-mercaptoethanol (Sigma‒Aldrich), penicillin‒streptomycin (Gibco), and 5% knockout serum replacement (KSR, Gibco). The optimized medium was N2B27 medium supplemented with numerous small molecules and cytokines, as follows: recombinant human LIF (10 ng/ml; Peprotech), CHIR99021 (1 µM; Gene Operation), (S)-(+)-dimethindene maleate (2 mM; Tocris) and minocycline hydrochloride (2 mM; Santa Cruz). IWR-endo-1 (1 mM; Selleckchem) and Y-27632 (2 mM; Peprotech) were included in some cases. ES cells were passaged routinely using 1 mg/mL type IV collagenase (Sigma) (traditional culture ESCs) and 0.05 trypsin (optimized culture ESCs) every 3–4 days. The haNSCs were passaged every 4 days with 0.05% trypsin. The haNSC medium contained D/F12 + 1% N2 supplement + 20 ng/mL basic fibroblast growth factor (bFGF, Peprotech) + 20 ng/ml epidermal growth factor (EGF, Peprotech). All cells were cultured in a 5% CO2 incubator at 37°C.
Neural differentiation of EXHPGES
Well-conditioned EXHPGES were selected for differentiation, as the state of haESCs plays a decisive role in neural differentiation.. EXHPGES were digested to single cells with 1 mg/ml 0.05% trypsin. Then, these single cells were placed into gelatine-precoated dishes for 30 minutes to allow the feeder cells to attach to the bottom of the dish. Single cells were suspended in EB medium in a suspension petri dish for 4 days to form embryoid bodies. During the first day of differentiation, we added 10 µM ROCKi to EB medium. On Day 4, the EB medium was replaced by neural induction medium (NIM) for another 2 days. On Day 6, we transferred the EB from the suspension petri dishes to laminin-precoated dishes overnight. The next day, EB paste was added to the bottom of the dish. We changed the NIM every other day. The growth of flat cells and an increasing number of slender small cells were observed in the centre of the implanted EB. After approximately 10 days in NIM, the central, small, lanky cells generated rosettes. Clusters were picked and expanded as described by Zhang et al. with slight optimizations [35–37]. In short, rosette clusters were attached to laminin-precoated Petri dishes in N2 medium containing human bFGF and EGF. Then, haNSCs were derived from haploid rosette clusters with continuous culture on Plo/Lam culture dishes in N2 medium with 20 ng/ml bFGF and 20 ng/ml EGF for three months. The differentiation efficiency from haploid rosette clusters to haNSCs under this condition was nearly 100% according to cell morphology evaluation.
Neuronal differentiation of haNSCs
Haploid neural stem cells (haNSCs) were examined for their neural differentiation potency. For differentiation into neurons, D/F12 medium was added as basal medium and supplemented with N2, B27 (Gibco), BDNF (20 ng/ml), glial-derived neurotrophic factor (20 ng/ml), 1 mm dibutyryl-cAMP and 200 nM ascorbic acid were added. Then, the haNSCs were replated on laminin-coated Petri plates at a density of 1×104 cells/cm2. The addition of 1% FBS (BI) to the primary medium was used to differentiate haNSCs into astrocytes and oligodendrocytes [36].
DNA Content Analysis of Cells
Human ESCs and NSCs were dissociated into single cells by treatment with 0.05% trypsin/EDTA at 37°C for approximately 3 min. Cells were fixed in 75% ethyl alcohol at 4°C at least 12 hours and stained with propidium iodide (PI) plus RNase A. Flow cytometry data were recorded by a BD Aria II system.
FACS sorting
For sorting of haploid cells, haESCs and haNSCs were disaggregated by 0.05% trypsin/EDTA, washed with Dulbecco’s phosphate-buffered saline and then incubated with 4 µg/ml Hoechst 33342 in a 37°C water bath for approximately 15 min. Subsequently, the haploid cells was collected using a BD FACSAria III for further culture. ROCKi (10 µM Y27632) was added before and after sorting.
RNA isolation & real-time PCR
Total RNA from all cells was extracted with TRIzol reagent (Invitrogen, CA, USA) and then treated with DNase I (Promega, WI, USA). cDNA was obtained by first-strand synthesis using 2 µg of RNA and the ReverTra Ace system for RT‒PCR (Toyobo, Japan).Real-time PCR was performed as described in our previous report. All reactions were repeated three times. Data are the mean ± s.d.
Immunocytochemistry
Immunostaining and AP staining of ESCs and NSCs were performed as described previously[38]. In short, cells were fixed in formaldehyde, rinsed with PBS, and permeabilized with Triton X-100 (Sigma). Then, incubated with blocking buffer. Rinsed with PBS, incubate with primary antibodies and secondary antibody (Abcam). After being rinsed with PBS, the cells were examined under a fluorescence microscope. The data represent three independent experiments.
Karyotype (G-banding) analysis
Cells were incubated with 0.2 µg/ml colchicine for 1 h (ESCs) or 2 h (NSCs). Then, the cells were disaggregated with 0.05% trypsin/EDTA and resuspended in 5 ml of culture medium. The cells were then harvested from the 5 ml suspension in an automatic cell harvesting apparatus (Sinochrome, Chromprep II) according to the set procedure. Briefly, 4.5 ml of preheated KCl (0.075 mM) was added at 37°C for 30 min. Hypotonic solution-treated cells were fixed in methanol:acetic acid (3:1 volume:volume) for 20 min at 37°C, and the fixation step was repeated three times. Then, they were dropped onto precleaned charged slides by an automatic production machine (Sinochrome, Chromprep AS). After being dried in air, the cells were stained with Metaphase Auto-Stainer (Sinochrome, Chromprep G). More than twenty metaphase spread G-bands were analysed by MetaClient, Ikaros.
Growth curve detection
Cells were plated onto 96-well plates at a density of 1000 cells per well. Ten microlitres of Cell Counting Kit-8 (CCK-8) medium (Beyotime) was added to each well on days 1, 2, 3, 4, and 5. The CCK-8 assays were performed as reported previously.
Global gene expression analysis
All the RNA-seq data were sequenced by a Chinese company (Novogene). Total RNA was extracted using TRIzol Reagent (Invitrogen). Following the manufacturer’s instructions, raw data were processed with the FASTX toolkit to remove noise and adaptors. The R heatmap function was used to perform hierarchical clustering.
PB transfection and splinkerette PCR
PB (PiggyBac Dual promoter PB513B-1, ABI) and PBase (Super PiggyBac Transposase PB200PA-1, ABI) were cotransfected into cells with Lipofectamine 3000 (Invitrogen). Puromycin at a concentration of 250 µg/ml was used to culture and screen the transfected cells. To characterize the sites of the PB element insertion, genomic DNA was digested with BstYI (Thermo) at 37°C for 16 h, and the products were self-ligated. Then, with the linked DNA as a template, PCR was performed with the primers PB-RF and PB-RR to detect the previously reported insertion sites. PCR was performed as previously reported. The first round was performed as follows: 98°C for 75 sec; 2 cycles of 98°C for 20 sec and 64°C for 20 sec; 30 cycles of 98°C for 20 sec, 58°C for 15 sec, and 72°C for 2 min; 72°C for 7 min; and a final hold of 4°C. The second round was conducted as follows: 98°C for 75 sec; 30 cycles of 98°C for 20 sec, 58°C for 15 sec, and 72°C for 90 sec; 72°C for 7 min; and a final hold of 4°C. Splinkerette PCR and deep sequencing were also performed to identify the integration sites. All primers used are listed in Table S3.
High-throughput sequencing analysis
After sequencing of the PB mutation library, adapters and PB tags were removed from the read data before mapping to the genome using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/commandline.html). HaSAPPy 18 was run to align the trimmed reads to the genome assembly from UCSC (mm10)[39]. We analysed the insertions for each gene with GENCODE[40] with the default parameters.
Statistical analysis
Student’s t test was used to evaluate the differences in gene expression levels among groups. All statistical analyses were performed using SPSS software 13.0 and Image-Pro Plus.