Human embryonic stem cells contribute to embryonic and extraembryonic lineages in mouse embryos upon inhibition of apoptosis

Human embryonic stem cells contribute to embryonic and extraembryonic lineages in mouse embryos upon inhibition of apoptosis

Primers targeting BCL2L1 were used to detect the contribution of BCL2L1overexpressing hESCs in chimeric placenta. M, marker.

Mice and ethical review
Mice were purchased from Beijing Vital River Company. All animal experiments were performed in compliance with the guidelines of the Institute of Zoology, Chinese Academy of Sciences. The cross-species chimeric experiments performed were reviewed and approved by the Ethics Committee of Institute of Zoology, Chinese Academy of Sciences, and followed the ethical guidelines for human embryonic stem cell research released by the International Society for Stem Cell Research (ISSCR).

Vector construction and virus package
For BCL2L1-2AdsRed-IRESNEO and BCL2-2AdsRed-IRESNEO vector construction, the BCL2L1 and BCL2 fragments were cloned from human cDNA library.

Derivation and cultivation of hESCs
All the reagents and media used for this study were purchased from Life Technologies Inc. unless otherwise mentioned. hESCs were derived from human blastocysts as previously reported. Established prime state hESCs and BCL2L1/BCL2-overexpression hESC lines were cultured with E8 medium on matrigel coated 6-well dishes. All of the stem cells were cultured at 37°C in a 5% CO2 incubator.
Medium were changed every other day.

Derivation of AAVS1-Knockin cell line
Well cultured prime state hESCs were digested by accutase and collected by centrifuge. 10 6 hESCs were electroporated with 10 μg AAVS1-CAG-EGFP vector and 10 μg sgRNA-CAS9 expression vector by use 4D-Nucleofector (Lonza) using P3 Primary Cell 4D-Nucleofector kit (Lonza). Transfected cells were seeded onto mitotically inactivated MEFs in E8 medium combined with Revita-cell supplement (ThermoFisher). In the next day, medium was changed into standerd hESCs culture medium as previously reported. Three days later, 0.5 μg/ml puro was added in culture medium and after selection for 2 weeks, GFP-positive and puro-resistant clones were picked and identified by PCR.

Embryoid body formation
For EBs formation, prime state hESCs were digested into single cells by accutase and were transferred to low-cell binding U-bottom 96-well plate (NUNC) in the medium containing GMEM (Sigma), 15% KnockOut serum replacement, 0.1 mM Glutamine, 0.1 mM NEAA, 0.1 mM b-mercaptoethanol. The medium was changed every other day.
After 5 days and 10 days, the EBs were collected and stored in Trizol reagent in -80°C.

Real-time PCR
Total RNA was isolated from freshly obtained cells using Trizol reagent. First strand cDNA was synthesized using MMLV reverse transcriptase (Promega) and oligo-dT (Promega) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Green Real-time PCR Master Mix (Toyobo) on Agilent Mx3005P. All primers used in the research are listed in Table S2.

Interspecies chimera generation
Mouse embryos were used as recipients for interspecies chimera generation.
Mouse 2-cell stage embryos were recovered from 1.5 days post coitus (d.p.c.) oviducts and cultured to 4-cell stage embryos with KOSM medium (Sigma). For interspecies chimera generation, anti-apoptotic gene transgenic hESCs were treated with or without 2 μg/ml DOX 24 hours before injection. Anti-apoptotic gene transgenic hESCs were digested into single cells using Accutase and suspended in culture medium.
Injections were performed with a micromanipulation system. After being cultured for about 24 h in KSOM medium (Sigma) with or without DOX treatment (2 μg/ml, Sigma), these manipulated 4-cell staged embryos developed to blastocysts. Chimeric blastocysts were transferred into the uteri of 2.5 d.p.c. pseudopregnant CD-1 mice.
Surrogate mothers were fed with DOX containing water (2 mg/ml) until the embryos developed to E6.5. The embryos or placentas were analyzed for EGFP expression, real-time PCR, immunofluorescence staining and fluorescence-activated cell sorting at E6.5, E8.5 and E10.5.

Immunofluorescence staining
Stained cells and embryos were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 in PBS for 10 min followed by blocking with 2% BSA (Sigma) in PBS. The cells were incubated with primary antibodies overnight at 4°C, followed by the use of secondary antibodies (Jackson Immuno Research) at room temperature for 1h. Finally, DNA was stained with 10 μg/ml Hoechst 33342 for 30 min.
E10.5 embryos and placentas were embedded in OCT (VWR), frozen and cryosectioned at 10 μm, then stained as described above. Images were captured with a Zeiss LSM780 Meta inverted confocal microscope. Antibodies against OCT4 (Santa The accession number for the RNA-seq expression values reported in this paper is GSE100862.

Statistical analysis
All data are presented as mean ± SEM. Each experiment included at least three independent samples. Statistical parameters including statistical analysis, statistical significance, and n value are reported in the Figure legends. Statistical analyses were performed using Prism Software (GraphPad). For statistical comparison, one-way ANOVA was employed. A value of p < 0.05 was considered significant.