Culture of metastable and ground-state mouse ES cells
Metastable ES cells were grown on 0.1% (w/v) gelatin-coated cell culture dishes in serum+ LIF media, comprising 10% (v/v) heat-inactivated FBS in GMEM (12.5g/litre w/v), NaHCO3 (32.7mM), Sodium Pyruvate (1mM), NEAA (0.1mM) and β-mercaptoethanol (0.1mM) and LIF. Ground state ES cells were cultured on 0.1% gelatine-coated tissue culture-treated dishes in N2B27, containing DMEM/F12 and Neurobasal medium in 1:1 (v/v) ratio, supplemented with N2 supplement (1X), B27 supplement (1X), NaHCO3 (32.7mM), Sodium Pyruvate (1mM), NEAA (0.1mM), and β- mercaptoethanol (0.1mM) along with recombinant hLIF (1000U/ml), CHIR99021 (3μM) and PD0325901 (1μM). TrypLE-EDTA was used for dissociation and passing once the confluence reached 70%.
Extended pluripotent stem cell culture
Metastable ES cells were converted into Extended pluripotent stem cells (EPSCs) when grown in either of two different culture regimes. First as described by Pentao Liu’s group33, where DMEM/F12 (Invitrogen), 20% (v/v) KnockOut Serum Replacement (KSR), NEAA (1X), β- mercaptoethanol (0.1mM) and hLIF 1000 U/ml supplemented with the following small-molecule inhibitors: CHIR99021 (3μM), PD0325901 (1μM), JNK Inhibitor VIII (4μM), SB203580 (10μM), A-419259 (0.3μM) and XAV939 (5μM). The second culture media as described by Hongkui Deng’s group33 where basal N2B27 media was supplemented with small molecules and cytokines as follows: 10 ng/ml recombinant hLIF, CHIR99021 (3µM), (S)-(+)-Dimethindenemaleate (2µM), Minocycline hydrochloride (2µM) and 5mg/ml BSA. Upon 70% confluency, EPSCs were passaged with TrypE-EDTA, and splitting was done in a 1:3 ratio.
Derivation of ESTS from ES cells and TSC from blastocyst
For the derivation of trophoblast stem cells (ESTS) from mESCs, E14Tg2a cells were cultured for 48hrs in SL media supplemented with CHIR99021 (3μM). Thereafter, cells were passaged and seeded onto culture plates as described by Ohinata et. al.22. Geltrex (1x) or 15µg/ml fibronectin solution in PBS was used to coat the tissue culture-treated plates at 37oC for 1 hour. SLCHIR-treated E14Tg2a cells were seeded onto these plates and cultured in CDM/FAXY media. CDM/FAXY media contained DMEM/F12: Neurobasal media (1:1 v/v), supplemented with N2 (1X) and B27 (1X) supplements, BSA (0.05% v/v), 1-thioglycerol (1.5 X 10-4M), recombinant human bFGF (25ng/ml), recombinant human Activin A (20 ng/ml), XAV939 (10 µM), and Y27632 (5µM). By day 6, colonies with TS-like morphology were surrounded by contaminating non-TS-like cells. Two methods were used to separate ESTS from the contaminating cells. First, by manually picking the colonies having TS-like morphology with a fine pipette and seeding them back into coated dish with FAXY media as a single cell suspension. In another method, CDM/FAXY media was removed, and the culture was treated with Accutase for 30sec or until the contaminating cells started to detach from the dish after gentle tapping. The dishes were washed with PBS to remove the detached cells, while the TS-like cells remained attached. This method utilises the deferential sensitivity of the TS-like cells and the contaminating cells to the Accutase treatment. Media was changed every 24hrs and clearing of contaminating cells was done after the cell density reached 60% confluency. The first cleaned ES-TSC plate was named ESTSP1 followed by multiple passages as P2-P24. ESTSP24 or later passages are hereinafter called ESTS. TSC were derived from blastocysts as described in Ohinata et. al.22.
Mice
CD1 and F1 (C57BL6/CBA) mice were obtained from Jackson laboratories. Animal model experiments were carried out following the Institutional Animal Ethics Committee (IAEC) of the CCMB's ethical standards and received approval from the Committee for Control and Supervision of Research on Animals (CPCSEA). For pseudo-pregnancy, Vasectomized F1 male mice were mated with CD1 female mice that were 8 to 11 weeks old and in the estrus cycle. All the experimental mice were kept in a 12-hour light/12-hour dark cycle with free access to food and water in a facility with controlled temperatures (18-22oC) and humidity (R.H. 40-70%).
Superovulation of mice
5 IU of PMSG (pregnant mares' serum gonadotrophin) was administered to female F1 (C57BL6/CBA) for superovulation. 5 IU of human chorionic gonadotrophin (HCG) was administered 48 hours after the initial injection and Day 0 was designated of and the subsequent instant mating with F1 male mice. After the vaginal plug was found the following day, 0.5 dpc zygotes were retrieved and cultivated in KSOM in an incubator that was humidified and maintained at 37oC.
Culture of mouse embryos
The upper portion of the oviduct (ampulla) was cut-off from the pregnant mice, and zygotes (0.5dpc) and cumulus mass were extracted from F1 (C57BL/6J X CBA). A prepared M2 medium with 0.3 mg/ml hyaluronidase was used to transfer the oviduct area containing the zygotes. Close to the location of the zygotes, the ampulla was torn using a 26-gauge needle. For a short period of time, all of the zygotes were incubated in M2 medium containing hyaluronidase. Zygotes were picked up using a pipette after the cumulus mass had fallen off and transferred to new M2 drops before being recorded as Day 1 and pre-equilibrated at 37oC with 5% CO2 in a humid incubator. Every 24 hours, embryos were transferred into fresh KSOM drops.
Immunosurgery of E3.5 blastocyst
The immunosurgery technique was used to separate the inner cell mass (ICM) and trophectoderm (TE) of mice E3.5 blastocyst37. The blastocysts were transferred to the M2 medium and washed twice in the M2 medium. Blastocysts were incubated with Rabbit anti-mouse antibody at a dilution of 1:10 (diluted in DMEM plus 10% FBS) for 15 minutes at 37°C in a CO2 incubator. Blastocysts were washed three times with M2 medium after the incubation. Blastocysts were transferred to a fresh drop containing native guinea pig serum complement at a dilution of 1:50 (diluted in DMEM plus 10% FBS), and incubated for 30 minutes at 37°C in a CO2 incubator. The blastocysts were then observed under a microscope to confirm the lysis of the TE cells. Zona pellucida was cleared with acid Tyrode treatment for 2 mins. The ICMs were collected carefully using a micropipette and transferred to a new dish with fresh M2 medium. Finally, the ICMs recovered were transferred to Geltrex coated cell culture plate containing FAXY media for EpiTS differentiation.
Injection of ESCs, CHIR-treated ESCs, and ESTS into an 8-cell embryo
GFP expressing ES (HGFP) (cell line pedigree chart in supplementary data) cells were cultured on SL, SLCHIR (CHIR, 3µM) for 16hrs. SLCHIR media was changed freshly 3hrs before injection. For ESTS transfer, GFP labelled ESTS cells which were grown in FAXY for 24 passages (ESTSP24) or later passages cells were trypsinized with TrypLE-EDTA, neutralized, washed, and resuspended as single cells in injection media. Injection media was freshly made and constituted of DMEM/F12 (Sigma) supplemented with 10% (v/v) heat-inactivated ES qualified serum, Glutamax (1X), NaHCO3 (32.7mM), and pH was adjusted to 7.2. Resuspended ESCs were microinjected into the 8-cell stage embryo in an injection drop containing 200µl of injection media in a 50mm glass-bottom dish. Leica DM IRB inverted microscope was used with an Eppendorf TransferMan NK2 micromanipulator. The outside and inside diameters of the holding pipette were 95–105μm and 20–25μm respectively; whereas the outside and inside diameters of the injection pipette were 18–20μm and 16–18μm respectively. 2-3 ESCs/ESTS cells were microinjected per embryo on a 50mm dish containing injection media on a microscopic stage maintained at 100C. A batch of 20 embryos was injected in an hour.
Cryosectioning of placental tissue and cytohistochemistry
Placental tissues were obtained after dissecting 12.5 dpc mice. Tissues were immediately fixed in 4% paraformaldehyde and kept overnight at 4oC. Tissues were washed thrice with PBS and transferred to 15% sucrose solution in PBS and kept at 4oC until the tissues were sunk at the bottom of the tube. The tissues were again transferred to 30% sucrose solution in PBS and kept at 4oC overnight followed by OCT media embedding and taken for cryosectioning. Sections of 15-micron thickness were taken on ProbeOn slides and followed for immunocytochemistry. The slides were dried at room temperature for 5mins and washed with PBS twice to remove the OCT. Sections were blocked and permeabilized in a blocking buffer containing PBS with 5% (w/v) BSA, and 0.3% (v/v) Triton-X 100 for 60 minutes at room temperature. Primary Antibodies were diluted in antibody dilution buffer (ADB) containing PBS with 1% (w/v) BSA, and 0.3% (v/v) Triton-X 100. Sections were incubated with primary antibodies for 4oC overnight. Sections were washed 3 times with PBS and incubated with secondary antibodies diluted at 1:1000 in ADB. Sections were washed with PBS and mounted with Vectashield (H-1200) containing DAPI.
Generation of CRISPR-based gene knock-out cell lines
The twin guide strategy was used for generating knock-out in ES cells as described in Kale et. al.32. The cell line was characterized by genotyping, qPCR, and western blotting analysis to confirm the intended genetic manipulation. The detailed characterization and pedigree of the cell lines used in this study are provided in supplementary data (supplementary appendix).
Generation of ES cells knock-in cell lines
TCMC cell line was generated by using CRISPR as described by Jana et al. (Jana, et al. 2019). TCMC-OGFP cell line was generated by nucleofecting 1µg supercoiled Oct4-IRES-eGFP-PGK-Neo (Addgene #48681) plasmid to 1 million TCMC using P3 primary cell 4D-Nucelofector X kit (Lonza). Nucleofected cells were plated and grown in SL media supplemented with G418 for 10 days. A single colony was picked up and cultured as replicas in 96 well format for genotyping. The detailed characterization and pedigree of the cell lines used in this study are provided in supplementary data (supplementary appendix).
Immunofluorescence staining
Cells were cultured in 2D culture in 24 wells for up to 70% confluency. Cells were washed thrice with 500µl of PBS and 500 µl of freshly prepared 4% paraformaldehyde (made in PBS) fixative was added to the plate and incubated at RT for 20 minutes. The fixative was removed and the plate was washed thrice with 1ml PBS. The specimen was blocked and permeabilized in a blocking buffer containing PBS with 5% (w/v) BSA, and 0.3% (v/v) Triton-X 100 for 60 minutes at room temperature. Primary Antibodies were diluted in antibody dilution buffer (ADB) containing PBS with 1% (w/v) BSA, and 0.3% (v/v) Triton-X100. Specimens were incubated with primary antibodies for 4oC overnight. Cells were washed 3 times with PBS and incubated with secondary antibodies diluted at 1:1000 in ADB. Cells were washed with PBS and mounted with Vectashield (H-1200) containing DAPI.
For staining 3D structures like embryos, they were first washed twice with PBS. Structures were fixed in 4% paraformaldehyde in PBS for 15 minutes and rinsed in PBS containing 3 mg/ml polyvinylpyrrolidone (PBS/PVP). Thereafter structures were permeabilised in PBS/PVP containing 0.25% Triton X-100 for 30 minutes. Blocked in blocking buffer, comprising PBS containing 5% BSA, 0.01% Tween 20 for 60 minutes. Primary antibodies were diluted with the appropriate antibody dilution as per the manufacturer protocol in PBS containing 1% BSA, and 0.01% Tween 20 and incubated at 4°C overnight. They were rinsed three times in blocking buffer for 5 minutes each and incubated with secondary antibodies diluted as 1:500 in PBS containing 1% BSA, 0.01% Tween 20, and incubated for 60 minutes at room temperature. Rinsed 3 times with PBS and stained for nuclei with DAPI (1µg/ml) prepared in PBS for 15 minutes at RT. Embryos were finally rinsed 3 times in PBS and images were acquired by confocal microscopy.
Real-time PCR analysis
The RNA was extracted from 1 million cells with TRIzol by manual method and quantified by Nanodrop (Thermo Scientific). One microgram of RNA was reversed and transcribed into cDNA by using SuperScript™ III First-Strand Synthesis System. The first strand synthesized cDNA was diluted 5 times and real-time PCR was set with power SYBR Green PCR master mix on the ABI 7900 HT. The PCR setup was as follows: Step 1: 95oC for 10 min, step 2: 95oC for 15 sec, step 3: 60oC for 30 sec, and Step 4: 72oC for 30-sec Steps 2-4 were repeated for 40 cycles. GAPDH was used as an internal control. The reactions were analyzed by the software (SDS 2.1) provided with the instrument. The primers used for real-time PCR are given in the resource table.
Western blot analysis
The cells were harvested by scraping them from the plates in PBS and collected by centrifugation. The cell pellet was washed twice with PBS and reconstituted in RIPA. RIPA buffer constituting 25mM Tris HCl (pH 8.0), 150mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, and Complete Protease Inhibitor Cocktail Tablets (Roche). The lysate was sonicated in Bioruptor (Diagenode) and centrifuged. The clear supernatant was transferred to the fresh tube. The protein samples were denatured in loading dye containing β-mercaptoethanol and resolved by SDS-PAGE. Resolved samples were then transferred onto a polyvinylidene difluoride (PVDF) membrane and blocked-in blocking buffer containing 5% non-fat milk in TBST. Primary antibody hybridisation was carried out overnight at 4oC in 3% non-fat milk in TBST. After incubation three washes were performed with TBST. The secondary antibody was diluted at 1:10,000 in 3% non-fat milk in TBST and incubated for an hour at RT. After incubation three washes were performed with TBST and visualized using enhanced chemiluminescence (ECL) detection kit (Thermo Scientific) and developed in Chemi doc MP (Biorad).
FACS analysis
70% confluent culture was made into single-cell suspension using TrypLE for 4 mins at 37oC. The cells were diluted in media and pelleted by spinning at 300g for 5 mins. The media was removed and around 1 million cells were resuspended in 300 µl of PBS containing 2% FBS. The cell samples were directly taken for analysis either in Gallios (Beckman Coulter B5-R1-V2) FACS analyzer or LSR Fortessa (BD) analyzer and data was recorded. The FACS data were analyzed using FlowJo vX.0.7
Bulk RNA-seq library preparation
Using the TRIzol reagent and the manufacturer's instructions, total RNA was extracted from ESCs, TSCs and ESTSCs. A total RNA of 1μg was used to generate libraries using of Illumina stranded total RNA prep with Ribo-Zero Plus library preparation kit (Illumina, 20040529). The Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Q32854) was used to measure library concentration, and different libraries were combined to create the final pool at an equimolar ratio. Using an Illumina NovaSeq 6000 instrument, libraries were sequenced for a read length of 151 bp reads and a read depth of approximately 30 million reads.
Bulk RNA-seq data analysis
Paired-end Bulk RNA sequencing was done for ESCs, TSCs and ESTSCs. The quality assessment of raw data was done by FastQC v0.11.9 and Illumina universal adapter content was removed using cutadapt v2.8. The filtered sequencing reads were mapped to the mouse reference genome mm10 and Gene counts were obtained by STAR_2.5.4b. The data showed an average of 81% uniquely mapped reads which were annotated with the Ensembl database. Transcripts Per Kilobase Million (TPKM) were calculated by the rsem-calculate-expression function of RSEM v1.3.3. DESeq2 v1.30.1 was used to get the differentially expressed genes based on the Bayes theorem. Genes showing expression of at least 50 in a row were retained for further analysis. Principal component analysis (PCA) and heatmap analysis were performed with the functions plotPCA and pheatmap in R. The visualization of differential expression of marker genes was performed on TPM counts after scaling and normalizing the read counts by row.
Single RNA-seq library preparation
Single-cell suspension was made from ESCs, TSCs and ESTSCs using the TrypLE-EDTA solution. Live cells were sorted as PI-negative populations using a FACS sorter. Approximately 5000 cells were loaded for Library preparation using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (10x genomics). Libraries were sequenced for a read length of 91 bp reads.
Single-cell RNA-seq data processing
Single-cell RNA-sequencing was performed using the 10X Genomics Chromium system and Illumina base call files (BCL) were demultiplexed using the mkfastq function of cellranger v6.0.2 which is specific to 10X libraries. Reads were aligned against mouse reference mm10 and filtered by the count pipeline to get the feature, barcode, and gene matrices. The R package Seurat v4.0.1 was used to analyze the feature-barcode matrix. The quality assessment of data was done by scater v1.18.6. Scrublet, a python tool, was used to calculate doublet scores and predictions. Cells with more than 500 detectable genes with a doublet score of <0.25 and expression of mitochondrial genes accounted for less than 5% of total expression were filtered from the dataset for further downstream analysis. The final dataset includes ESC, ESTS, and TSC derived from blastocysts. Samples were integrated using the merge function in the Seurat suite. Normalization and variance stabilization was done by sctransform and variations due to genes were regressed out. Cells were clustered with the FindClusters function with a resolution of 0.5 and visualization was done using the RunUMAP function.
Data and materials availability:
All the cell lines and plasmid constructs used in this study will be made available against and email request and Material Transfer Agreement (MTA). Raw and processed transcriptome data is deposited in NCBI GEO and is available under the accession GSE219001. The code used for analysis and visualization of scRNAseq data is available at https: https://github.com/SowpatiLab/ES-TSCs
Statistical analysis
Statistical analysis was done by using a two-tailed paired student t-test. The representation of data is in the form of means+/-SDM. The was calculated for more than three independent experiments P value<0.05 is considered statistically significant. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001.