Human fetal samples
All prenatal gonads were obtained from the University of Washington Birth Defects Research Laboratory (BDRL), under the regulatory oversight of the University of Washington IRB approved Human Subjects protocol combined with a Certificate of Confidentiality from the Federal Government. All consented material was donated anonymously and carried no personal identifiers, therefore the use of the de-identified fetal tissue at UCLA was deemed exempt by the UCLA IRB under 45 CRF 46.102(f). Developmental age was documented by BDRL as days post fertilization using prenatal intakes, foot length, Streeter’s Stages and crown-rump length. All prenatal gonads documented with birth defect or chromosomal abnormality were excluded from this study.
Human ESC culture
The hESC lines in this study are as follows: UCLA1 (46, XX), UCLA2 (46, XY)34, UCLA8 (46, XX)22, and H1 OCT4-GFP (46, XY)12. All hESCs were cultured on mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) and split every 7 days using Collagenase type IV (GIBCO, 17104-019). hESC media was comprised of 20% knockout serum replacement (KSR) (GIBCO, 10828-028), 100mM L-Glutamine (GIBCO,25030-081), 1x MEM Non-Essential Amino Acids (NEAA) (GIBCO, 11140-050), 55mM 2-Mercaptoethanol (GIBCO, 21985-023), 10ng/mL recombinant human FGF basic (Proteintech HZ1285), 1x Penicillin-Streptomycin (GIBCO, 15140-122), and 50ng/mL pri-mocin (InvivoGen, ant-pm-2) in DMEM/F12 media (GIBCO, 11330-032). All hESC lines used in this study are registered with the National Institute of Health Human Embryonic Stem Cell Registry and are available for research use with NIH funds. hESCs used in this study were routinely tested for mycoplasma (Lonza, LT07-418). All experiments were approved by the UCLA Embryonic Stem Cell Research Oversight Committee.
Undifferentiated hESCs, iMeLCs, D4 hPGCLCs (EPCAM/ITGA6), and D4 somatic cells (the EPCAM/ITGA6 negative cells) were re-suspended in 350 uL RLT buffer (QIAGEN) and RNA was extracted using RNeasy micro kit (QIAGEN). RNA was converted to cDNA using SuperScript® II Reverse Transcriptase (Invitrogen). Real-time quantitative PCR was performed using TaqMan® Universal PCR Master Mix (Applied Biosystems), and with Taqman probes detecting expression of GAPDH, TFAP2C, SOX17, PRDM1, KLF4, and TFCP2L1. Expression levels for genes of interest were normalized to housekeeping gene GAPDH in each cell type. To quantify relative expression in each cell type, expression levels was normalized to the expression level of hESCs. hPGCLCs were compared to hESCs in biological triplicate, and to iMeLCs and somatic cells in duplicate. P-values were calculated using two-tailed student T test.
Aggregates collected at D4, and human fetal tissue samples were fixed in 4% PFA for 1 hour, washed twice for 15 minutes in PBS, stained with hemotoxylin, and mounted in histogel (Thermo Scientific). Samples were embedded into paraffin blocks and cut onto slides in 5 um- thick sections. Slides were deparaffinized and rehydrated through a series of xlene and ethanol series. For antigen retrieval, slides were heated to 95C in Tris-EDTA solution (10 mM Tris Base, 1 mM EDTA solution, .05% Tween-20, pH9.0). Sections were permeabilized (.05% Triton-100 in PBS) for 20 minutes and blocked in PBS containing 10% normal donkey serum for 1 hour. Primary antibodies incubated overnight at 4C. Antibodies included anti-TFAP2C (sc12762; 1:100), anti-SOX17 (GT15094; 1:100), anti-Blimp1 (9115S; 1:100), anti-KLF4 (AF3640; 1:100), anti-TFCP2L1 (AF5726; 1:100), and (cKIT A405 1:100). The next day, slides were washed, blocked for an additional 30 minutes, and stained with secondary antibody for 1 hour in their corresponding species-specific secondary antibody. Secondary antibodies included donkey anti-mouse 488 IgG (715-546-150; 1:200), donkey anti-mouse 594 IgG (A21447; 1:200), donkey anti-rabbit 488 IgG (711-545-152; 1:200), donkey anti-rabbit 594 IgG (711-585-152; 1:200), donkey anti-rabbit 647 IgG (711-605-152; 1:200), donkey anti-goat 488 IgG (705-546-147; 1:200), donkey anti-goat 594 IgG KLF4, TFCP2L1 (705-586-147; 1:200), donkey anti-goat 647 IgG KLF4, TFCP2L1 (A21447; 1:200). Dapi (xxx; 1:1000) was added during secondary antibody incubation and samples were mounted in ProLong Gold antifade reagent (Invitrogen).
hPGCLCs at D4C10 or D4C21 on chamber slides, and 5iLF/A cells split onto coverslips in culture at P3 were washed and fixed in 4% PFA for 10 minutes. Cells were washed, permeabilized, blocked, and stained as described above. Primary antibodies were anti-KLF17 (042649 1:200), and anti-OCT4 (sc-5279 1:100). Secondary antibodies were incubated for 30 minutes.
For Edu analysis, cells in culture were incubated with Edu for 4 hours fixed, and detected using Click-iT™ Edu Cell Proliferation Kit for Imaging, Alexa Flour™ 488 dye before permeabilization.
hPGCLCs in aggregates were quantified in IMARIS 8.1 (Bitplane). For KLF4 and quantification, we counted the percentage of cells that were KLF4+ in the TFCP2C, PRDM1 double-positive hPGCLC population. This was repeated in 3 cell lines. In human fetal samples, we counted the total CKIT positive hPGCs, and then identified how many of these were KLF4 or TFCP2L1 positive. This was repeated in 6 samples for male and female each.
To identify the number of naive colonies during reversion at P3 in the mutant compared to control, 20 representative images of each sample were acquired at 10X zoom. Oct4 positive colonies were scored as full KLF17 positive, some KLF17 positive, and KLF17 negative. Counts for each category was normalized to the control.
In extended culture, total hPGCLC colonies in each well were counted and normalized to the number of cells plated down for each sample. Each was performed with 2 technical replicates. For Edu quantification, the number of Edu positive cells in each hPGCLC colony was counted and Edu percentage of each hPGCLC colony was compared between the mutant and control-dervied hPGCLCs. Error bars on graphs indicated standard error.
hESC mutants made by CRISPR/Cas9
To make null-mutations for KLF4 and TFCP2L1, pairs of gRNAs were designed to target the functionally important, most N-terminus coding region of each gene. Guides were designed using https://zlab.bio/guide-design-resources, and cloned into PX459 vector35. Pairs of guides were designed approximately at a 3 kB distance from each other in the genome. 4-6 days before nucleofection, UCLA1 or H1-OCT4-GFP hESCs were purified from MEFs and transferred to matrigel (BD) in mTeSR media (stemcell tech). At 70% confluence, cells were electrophorated with 4 ug of each gRNA pair was using P3 Primary Cell 4D-Nucleofector® X Kit according to the manufacturer’s instructions (Lonza, V4XP-3024). 1 day following recovery, cells were dissociated with Accutase and replated on DR4 MEFs in a 6-well plate. Cells were treated with puromycin at a concentration of .35 ug/mL for 1 day. Once colonies emerge after 6-8 days, they are dissociated and plated at 10k and 50k densities in 10 cm plates. 48 colonies were picked when at the desired density afte 10 days. Colonies were split in half after 4 days. To determine homozygous mutants, we genotyped genomic DNA and chose colonies with the expected shorter band. Genotyping primers and gRNA sequences are listed in supplementary table 1. To confirm bi-allelic mutantions, mutant bands were cloned into Blunt-PCR-Cloning vector using Zero Blunt PCR Cloning Kit (TheroFisher, K270020). 5-10 colonies from each band were picked and sequenced.
hPGCLCs were induced as described previously22. 1 hour before plating, 12-well plates are treated with human plasma fibronection (Invitrogen). hESCs were washed and dissociated in 0.05% trypsin for 5 minutes, and quenched with MEF media. The MEFs were removed by plating in 10-cm cell culture dishes, twice for 5 minutes each. Purified hESCs were spun, filtered with 100um filter, and seeded at a density of 200k per 12-well in iMeLC media including 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 50ng/mL Activin A (Peprotech, AF-120-14E), 3mM CHIR99021 (Stemgent, 04-0004), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035). After 24 hours, cells were dissociated with trypsin, inactivated with trypsin inhibitor (Sigma), resuspended in PGLCC media, and plated in ultra-low cell attachment U-bottom 96-well plates (Corning) at a density of 3k cells/well. hPGCLC media is comprised of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 10ng/mL human LIF (Millipore, LIF1005), 200ng/mL hu-man BMP4 (R&D systems, 314-BP), 50ng/mL human EGF (R&D systems, 236-EG), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035).
Flow cytometry and fluorescence activated cell sorting
D4 aggregates were collected and dissociated with .05% trypsin for 10 minutes. The dissociated cells were stained with conjugated cell-surface antibodies for at least 15 minutes. Antibodies included anti-ITA6-BV421 (BioLegend, 313624 1:60), anti-EPCAM-488 (BioLegend 324210; 1:60) for UCLA-derived lines and anti-ITA6-488 (BioLegend, 313608 1:60), anti-EPCAM-APC (BioLegend 324208; 1:60) for H1-derived lines. After at least 15 minutes, cells were washed with FACS buffer (1% BSA in PBS), resuspended in FACS buffer with 7AAD (BD PharMingen 559925; 1:40). Single-cell suspensions of hESCs were used as single-color compensation controls and evaluated each with 7AAD, anti-ITA6-BC421, and anti-EPCAM-488 for UCLA1-derived lines and anti-ITA6-488, anti-EPCAM-APC, and unstained GFP-OCT4-expressing cells for the H1-OCT4-GFP derived lines. For gating controls, fluorescence-minus-one controls were made against each fluorophore, staining the residual cells from the aggregate supernatant with each antibody, minus its respective control. Gating was then established based on the absence of signal in the cell population of interest. Double-positive ITGA6, EPCAM cells were analyzed using an ARIA-H Fluorescence Activated Cell Sorter and sorted into either 350 uL of FR10 media or RLT buffer. Analysis was performed using FlowJo version 10.
For flow cytometry, cells in 5iLF/A at passage 3 were dissociated using accutase, passed through a 40um strainer (BD) and resuspended in FACS buffer at equal cell numbers. Conjugated antibodies including anti-CD75-APC (ThermoFisher 50-0759-41; 1:20), anti-CD24-BV421 (BD 562789 1:40), anti-CD90.2-APC-Cy7 (BioLegend 105327 1:20), and fixable live-dead-APC-Cy7 (Fisher 50-169-66) were diluted in staining buffer (BD 563794) and used to resuspend cell pellet, staining in the dark for at least 15 minutes. Live, non-mouse cells were gated and analyzed for their percentage of CD75 positive, CD24 negative populations. Analyses was performed on an LSR Fortessa cytometer.
Primed to Naive Reversion
Cells were reverted to the naive ground state in 5iLF/A as described previously6,26. At day 7, primed hESCs were dissociated into single cells with accutase and re-plated on MEFs in hESC media with Y27632 (Stemgent, 04-0012-10) at a density of 200k cells/well per 6-well plate. After one day, media was changed to 5iLAF media including a 50/50 mixture of DMEM/F12 (Gibco 11320-033) and Neurobasal (Gibco 21103-049), 1X N2 (Gibco 17502-048), 1X B27 (17504-044), 20 ng/mL rhLIF (Millipore LIF1005), 1 mM GlutaMAX (Gibco 35050-061), 1% NEAA (Gibco 11140-050), .1 mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin (Gibco 15140-122), 50 ug/mL BSA (Gibco A10008-01), 1 mM PD0325901 (Stemgent 04-006-02), 1 mM IM-12 (BML-WN102-0005), .05 mM SB590885 (R&D 2650/10), 1 uM WH-4-023 (A Chemtek H620061), 10 uM Y-27632, 20 ng/mL Activin A (Peprotech AF-120-14E), 8 ng/mL FGF2 (Proteintech HZ1285), .50% KSR (Gibco 10828-028), and 1X primocin (Invitrogen (ant-pm-2). Media is changed daily and cells are passaged every 5 days at a ratio between 1:1 and 1:3 until robust colonies emerge. For quantification, 20 representative images were taken on the day of each passage 2-4. Colonies were scored as naive, primed, or intermediate. The mutant count was normalized to its corresponding control.
RNA sequencing library preparation and data analysis
Total RNA was extracted from H1-OCT4-GFP sorted D4 hPGCLCs using RNeasy micro kit (Qiagen 74004). Total RNA was reverse transcribed and cDNA was amplified using Nugen RNA-Seq System V2 (Nugen, 7102-32). DNA was extracted using MinElute PCR purification kit (Qiagen) and quantified using the Qubit dsDNA High-Sensitivity Kit (Life Technologies). Amplified cDNA was fragmented using Covaris S220 Focused-ultrasonicator. RNA-sequencing libraries were generated using Nugen Rapid Library Systems (Nugen 0320-32). Libraries were subjected to single-end 125 bp sequening on HiSeq4000 with 6 indexed libraries per lane.
RNA sequencing analysis
Raw reads in qseq format obtained from the sequencer were first converted to fastq files with a customized perl script. Read quality was evaluated with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). High-quality reads were aligned to the hg19 human reference genome using Tophat (v 2.0.13) by using “-no-coverage-search” option, allowing up to two mismatches, and only keeping unique reads. The number of unique mappable reads was quantified by HTseq (v 0.5.4) under default parameters. Expression levels were determined by RPKM (reads per kilobase of exons per million aligned reads) in R using customized scripts. For RNAseq of published datasets, GSE7697022 and GSE9312618, processed data of the raw read counts of each gene was utilized, with the same downstream analysis.
D4 hPGCLCs were cultured (C) for an additional 10 or 21 days as described previously27. Sorted hPGCLCs were plated at densities ranging 200-3000 cells in either a chamber well (D4C10) or 24-well (D4C21) in FR10 media. FR10 medium36 contains 10% KSR, 2.5% FBS (Thermo Fisher Scientific, SH3007003), 1× NEAA (Gibco, 11140-050), 1 mM sodium pyruvate (Gibco, 11360070), 2 mM L-glutamine (Gibco, 25030081), 0.1 mM 2-mercaptoethanol (Gibco, 21985-023), 1× penicillin streptomycin (Gibco, 15140-122), 100 ng/mL SCF (PeproTech, 250-03), 10 μM forskolin (Sigma, F6886), 10 μM rolipram (Sigma, R6520), and 50 ng/mL primocin in Glasgow's MEM (Gibco, 11710-035). For D4C21, hPGCLCs were dissociated at D4C10 using .05% trypsin for 3 minutes. Cells are spun down at 1.6 rpm for 5 minutes, carefully resuspended, and plated at a 1:2 ratio in chamber well. Media was replaced daily until readout.