Distinct requirements for energy metabolism in mouse primordial germ cells and
their reprogramming to embryonic germ cells
Yohei Hayashi, Kei Otsuka, Masayuki Ebina, Kaori Igarashi, Asuka Takehara, Mitsuyo Matsumoto,
Akio Kanai, Kazuhiko Igarashi, Tomoyoshi Soga, and Yasuhisa Matsui
Supporting Appendix
1 www.pnas.org/cgi/doi/10.1073/pnas.1620915114
SI Materials and Methods
Data reporting. No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
Animals. MCH and C57BL/6 mice were purchased from Japan SLC. Oct4-deltaPE-GFP transgenic mice (1) were maintained in a C57BL/6J genetic background. The mice were kept and bred in an environmentally controlled and specific pathogen-free facility, the Animal Unit of the Institute of
Development, Aging and Cancer (Tohoku University), according to the guidelines for experimental animals defined by the facility. Animal protocols were reviewed and approved by the Tohoku
University Animal Studies Committee. Noon on the day of the plug was defined as embryonic day
(E) 0.5. E13.5, E12.5 and E11.5 embryos were obtained from female MCH mice mated with male
Oct4-deltaPE-GFP transgenic mice. Embryos were collected and dissected in Dulbecco’s modified
Eagle medium (DMEM, Gibco 11965-092) containing 10% fetal bovine serum (FBS). The genital ridges of male embryos were dissected.
Flow cytometry. The genital ridges containing PGCs from Oct4-deltaPE-GFP transgenic mice, prepared as described above, were incubated with 1.2 mg/ml collagenase (SIGMA C0130) in PBS containing 10 % FBS for 1 h at 37 oC. To prepare single-cell suspensions for flow cytometry, cells within the samples were dissociated by pipetting, and samples were filtered through a 40 µm pore nylon mesh (BD falcon 352340). A Bio-Rad S3e cell sorter was used to sort and collect viable PGCs with intense Oct4-deltaPE-GFP expression (~ 1 × 105 cells/sorting) and Somas without Oct4- deltaPE-GFP expression. It takes about 30 minutes for a sorting and we have checked the high survival rate (> 93 %) of each cell type immediately after sorting (Fig. S1A). For the metabolomic analysis, sorted cells were immediately treated for metabolite extraction as described below. For the proteomic analyses, sorted cells were washed with PBS, removed supernatant and stored at -80 oC. 2
Cells from ~5 times sorting were suspended to Cell lysis buffer for whole cell extract [20 mM
HEPES (pH = 7.9), 10 % Glycerol, 400 mM KCl, 1 mM EDTA, 1 mM MgCl2, 0.1 % NP-40, 0.5 mM DTT, and 1 × protease inhibitor cocktail (Roche 04 693 132 001)].
ESC culture. Vasa-RFP (VR15) ESCs (2, 3) were cultured in KnockOut DMEM (Gibco 10829-018) supplemented with 15 % FBS, 4 mM L-glutamine (Gibco 25030081), 0.01 mM nonessential amino acids (Gibco 11140-050), 0.1 mM b-mercaptoethanol (SIGMA M3148), 1,000 U/ml LIF (ESGRO
Millipore ESG1107) on mouse embryonic fibroblasts inactivated with mitomycin C (SIGMA
M4287). A Bio-Rad S3e cell sorter was used to sort and collect viable VR15 ESCs after 3 days in culture.
Metabolite extraction. The sorted E13.5 male PGCs, Somas and VR15 ESCs (sorted, see “ESC culture”) (~ 1 × 105 cells / sample) were washed twice with 5 % mannitol. Add 1 ml of MeOH containing 2.5 µM each of three IS1s [L-methionine sulfone (Wako 502-76641), 2-(N- morpholino)ethanesulfonic acid (MES, Dojindo 349-01623), D-camphor-10-sulfonic acid (CSA,
Wako 037-01032)]. Leave at rest for 10 min, vortex, and transfer 400 µl to new tube. Add 400 µl of
o CHCl3 and 200 µl of Milli-Q water and mix well. Centrifuge at 10,000 g for 3 min at 4 C, and transfer 400 µl of aqueous layer to an HMT 5 kDa ultrafiltration tube [UltrafreeMC-PLHCC 250 / pk for Metabolome Analysis (UFC3LCCNB-HMT)]. Centrifuge at 9,100 g for 2 h at 20 oC, collect its filtrate and store at -80 oC. Put together the filtrated cell extract from approximately 5 x 105 cells for one specimen of each cell type and dry them using an evacuated centrifuge for 2 h at 40 oC. Add 25
µl of Milli-Q water containing 200 µM each of two IS2s [3-aminopyrrolidine (Aldrich 404624) and trimesate (Wako 206-03641)] for CE-MS analysis. Collected three specimens were then analyzed as three biological replicates.
3
Mass spectrometry for metabolome. The concentrations of all the charged metabolites in samples were measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS, Agilent
Technologies, Santa Clara, CA) using the methods developed by the authors (4-6). Briefly, to analyze cationic compounds, a fused silica capillary (50 µm i.d. × 97 cm) was used with 1 M formic acid as the electrolyte (7). Methanol/water (50% v/v) containing 0.1 µM hexakis(2,2- difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µl/min. ESI-TOFMS was performed in positive ion mode, and the capillary voltage was set at 4 kV. Automatic recalibration of each acquired spectrum was achieved using the masses of the reference standards ([13C isotopic ion of a protonated methanol dimer (2MeOH+H)]+, m/z 66.0632) and ([hexakis(2,2- difluoroethoxy)phosphazene +H]+, m/z 622.0290). To identify metabolites, relative migration times of all peaks were calculated by normalization to the reference compound 3-aminopyrrolidine. The metabolites were identified by comparing their m/z values and relative migration times to the metabolite standards. Quantification was performed by comparing their peak areas to calibration curves generated using internal standardization techniques with L-methionine sulfone. The other conditions were identical to those described previously (5). To analyze anionic metabolites, a commercially available COSMO(+) (chemically coated with cationic polymer) capillary (50 µm i.d. x 105 cm) (Nacalai Tesque, Kyoto, Japan) was used with a 50 mM ammonium acetate solution (pH
8.5) as the electrolyte. Methanol/5 mM ammonium acetate (50% v/v) containing 0.1 µM hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µl/min. ESI-
TOFMS was performed in negative ion mode, and the capillary voltage was set at 3.5 kV. For anion analysis, trimesate and CAS were used as the reference and the internal standards, respectively. The other conditions were identical to those described previously (5).
In-Solution Digestion for proteome. Whole cell extracts (5 µg, 3 biological replicates) were diluted over 10-folds with 50 mM NH4HCO3 to final volume of 90 µl. Subsequently, 15 µl of 100 mM DTT
4
(in water) was added followed by incubation for 30 min at 56 oC. Reduced cysteine residues were alkylated by adding 15 µl of 200 mM iodoacetamide (in water) and incubation for 30 min at room temperature in the dark. For in-solution digestion, 1 µg of trypsin (Promega) was added, and samples were incubated overnight at 37 oC. The digest reaction was stopped by adding 3 µl of TFA. Digested peptides were purified with C18 Spin Columns (Thermo Fisher Scientific), dried through vacuum centrifugation and dissolved in 50 µl loading solution [5 % acetonitrile contained 0.5 % TFA].
NanoLC-MS/MS analysis for proteome. Tryptic peptides (10 µl) were loaded on an Easy-nLC
1000 system (Thermo Fisher Scientific) connected with reversed phase C18 columns (Trap column:
Acclaim PepMap 100, 75 µm × 20 mm, Separation column: PepMap RSLC, 75 µm × 250 mm;
Thermo Fisher Scientific). Peptides were eluted with gradient generated by solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in acetonitrile) as followed: 5-23 % B in 180 min at a flow rate of 150 nl/min, 35-90 % B in 5 min at a flow rate of 175 nl/min, maintained at 90 % B in 5 min at a flow rate of 200 nl/min. Peptides were then ionized and analyzed with Orbitrap Elite
(Thermo Fisher Scientific). Full scan MS spectra (from m/z 350 to 2000) were acquired in the
Orbitrap with a resolution 60,000 at m/z 400 with using lock mass option (m/z at 391.284290 and
445.120030), followed by MS/MS fragmentation in the linear ion trap with normalized collision energy of 30 % against 20 most intense ions with +2 or more positive charges. Precursor ions selected for fragmentation once were excluded from selection for 30 s.
Data Processing for proteome. MS/MS data were analyzed with Proteome Discoverer 1.4 (Mascot and Sequest HT) according to manufacturer’s instruction and searched against mouse uniprot protein database for protein identification. For semi-quantification of each protein, the node ‘Precursor Ions
Area Detector’ was used and calculated area values of each protein peak were compared among
E13.5 male PGCs, Somas and ESCs (Dataset S2). Up to two missed cleavages were allowed.
Precursor and fragment mass tolerance were set to 10 ppm and 0.4 Da, respectively. Variable 5
modifications were oxidation of methionine and deamination of asparagine or glutamine, Static modification was carbamidomethylation of cysteine. The obtained sequences were filtered and validated taking into account false discovery rate (FDR) < 5 %.
Analyses of metabolomic and proteomic data. We used MetaboAnalyst 3.0
(http://www.metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml) for statistical analyses of metabolomic and proteomic data (8). In data processing, features with > 50% missing values were removed and remaining missing values were replaced by a half of the minimum positive value in the original data (default configuration). The processed data were normalized using auto scaling method
(mean-centered and divided by the standard deviation of each variable). Statistical differences were calculated using Student’s t-test or one way ANOVA. P < 0.05 was considered as statistically significant differences. The statistically-different features were then classified with K-mean clustering method. Each protein cluster was functionally annotated using the Database for
Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/,
Classification stringency: medium) (9).
Metabolic flux analysis. Seahorse XF24 Analyzer was used to measure oxygen consumption rate and extracellular acidification rate of E13.5 male and female PGCs and Somas, E11.5 PGCs, VR15
ESCs and PGCLCs on day4 in culture. The sorted cells were resuspended with DMEM containing 10
o % FBS and 1 mM sodium pyruvate, and incubated for about 1 h at 37 C, 5 % CO2. Centrifuge at
1,000 rpm for 5 min at 4 oC and remove supernatant. Resuspend the cells with XF running medium
[XF Base Medium (Seahorse Bioscience 102353-100), 25 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate (Gibco 11360-070)]. Cells were plated in XF24 Cell Culture Microplates
(Seahorse Bioscience 100777-004) at a density of 8 - 24 x 104 cells per well and incubated for more than 30 min at 37 oC. Cells were treated with 0.5 µM oligomycin (an ATP synthase inhibitor,
Alomone labs O-500), 1 µM FCCP (an uncoupler of OXPHOS, SIGMA C2920) and 1 µM rotenone 6
(SIGMA R8875) + 1 µM antimycin (SIGMA A8764) (known ETC inhibitors) and measured following the manufacturer’s instruction (Table S3).
Immunostaining. Immunostaining were performed using genital ridges (E13.5 male, female) from
Oct4-deltaPE-GFP transgenic mice fixed with 2 % paraformaldehyde for 3 h at 4 oC and embedded with Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek 4583). The embedded samples were sectioned using cryomicrotome CM3050S (Leica) with a section thickness of 10 µm.
The sectioned samples were permeabilized in 1 % Triton X-100 in PBS for 15 min at room temperature, and blocked in 5 % bovine serum albumin (BSA) and 1 % Triton X-100 in PBS for 1 h at room temperature. The sections were then incubated with the primary antibodies diluted by 1 %
BSA and 0.1 % Triton X-100 in PBS overnight at 4 oC, and were incubated with the secondary antibodies in the same buffer with 1 µg/ml DAPI for 2 h at 4 oC. Samples were washed for 5 min × 3 by 0.1 % Triton X-100 in PBS after the primary and the secondary antibody treatments. Samples were mounted with VECTASHIELD (VECTOR H-1000) and observed with confocal laser scan microscope TCS SP8 (Leica). PGCs were detected as Oct4-deltaPE-GFP-positive cells. The Alexa-
568 fluorescence intensities of each single cell were measured from the obtained image using the histogram option of LAS X software (Leica) and average intensities of PGCs and Somas were calculated. The primary antibodies were: NDUFB9 (abcam ab200198, 1:250), ATP5I (mouse
ATP5K, Proteintech 16483-1-AP, 1:50) and GSR (abcam ab16801, 1:1000). Goat anti-rabbit secondary antibody, Alexa Fluor 568 (Thermo Fischer Scientific A11011) was used at 1:500 dilution.
Culture of PGCs. PGC culture for reprogramming was carried out as described previously (10) with some modifications. The sorted E12.5 PGCs were cultured on a feeder layer of Sl/Sl4-m220 cells
(10) pre-treated with mitomycin C in 4-well tissue culture dishes with EG medium, which was based on the previously reported germline stem cell (GSC) culture medium (11) with modifications
[StemPro34 SFM (Gibco 10640-019) containing StemPro34 Nutrient, 100 µg/ml transferrin (SIGMA 7
T8158), 2 mM L-glutamine, 25 µg/ml insulin (SIGMA I2643), 50 µM b-mercaptoethanol, 20 ng/ml
EGF (SIGMA E4127), 25 ng/ml human bFGF (SIGMA F0291), 1,000 U/ml LIF, 100 U/ml penicillin-streptomycin (SIGMA P7359) and 10 % knockout serum replacement (KSR, Gibco
10828-028)]. In some experiments, 1 mM 2-deoxy-D-glucose (2DG, SIGMA D8375) or 0.12 µM rotenone was added. In the experiment for examining glucose-concentration dependency, glucose- free DMEM (Gibco A14430-01) was used instead of StemPro34. After 8-9 days in culture, staining for alkaline phosphatase activity was used to identify EGC colonies, as described previously (12, 13).
The efficiency of EGC formation was determined as a ratio of the number of EGC colonies to every
100 sorted PGCs that were seeded in a culture well. The survival rate of E12.5 PGCs was determined by counting the Oct4-deltaPE-GFP or ALP-positive cells. TdT-mediated dUTP-X nick end labeling
(TUNEL) staining for apoptosis detection in E12.5 PGCs was performed using In Situ Cell Death
Detection Kit, TMR red ver. 11 (Roche 12 156 792 910) according to manufacturer’s instruction after 16 h in culture. Feeder-free EG derivation was performed as reported previously (14). E11.5
PGCs were sorted and cultured for 48 h on 20 µg/ml fibronectin-coated (human plasma fibronectin,
Millipore FC010) 4-well tissue culture dishes in N2B27 medium with 3 µM CHIR99021 (CH, a
GSK3 inhibitor), 1,000 U/ml LIF, 25 nM human bFGF (SIGMA F0291), 2 µM all-trans retinoic acid
(SIGMA R2625), 10 µM forskolin (SIGMA F6886), and 100 ng/ml SCF (R&D 455-MC-010). After
48 h, cultures were transfered to N2B27 with 2i/LIF (3 µM CH, 1 µM PD325901 and 1,000 U/ml
LIF) by daily half-medium changes and GFP-positive colonies were counted on day 7~8.
PGCLC induction. PGCLC induction was performed as described previously (15). In this system, it was used ESCs with the reporter transgenes inserted fluorescent protein genes in the regulatory regions of PGC-specific genes, Prdm1 (also known as Blimp1) and Developmental Pluripotency
Associated 3 (Dppa3, also known as Stella) (Blimp1-mVenus-Stella-ECFP: BVSC), which were kindly provided from Dr. Mitinori Saitou. 2,000 (for metabolic perturbation) or 20,000 (for flux
8
analyzer) cells of EpiLCs were cultured in a 96U bottom plate (Thermo Fisher Scientific 174925) in
Glasgow’s Modified Eagle’s Medium (GMEM, Gibco 11710-035) supplemented with 15 % KSR,
0.1 mM nonessential amino acid, 1 mM sodium pyruvate, 2 mM L-glutamine, 180 µM b- mercaptoethanol with or without cytokines [500 ng/ml BMP4 (R&D 314-BP-050), 500 ng/ml
BMP8a (R&D 1073-BP-010), 100 ng/ml SCF (R&D 455-MC-010), 1,000 U/ml LIF and 50 ng/ml
EGF]. In some experiments, 1 mM 2DG or 0.03 µM rotenone was added. In the experiment for examining glucose-concentration dependency, glucose-free DMEM was used instead of GMEM.
Induced BV-positive PGCLCs were sorted on day 2 or day 4 post induction with a Bio-Rad S3e cell sorter and suspended to buffer RLT containing 1 % b-mercaptoethanol for total RNA extraction.
RNA preparation and reverse transcription real-time PCR. Total RNA samples were purified using RNeasy Micro Kit (QIAGEN 74004) according to the manufacturer’s instruction. RNAs were reverse-transcribed using SuperScript III (Invitrogen 18080044) and random primers (Promega
C118A). Expression levels of genes were quantified using the SYBR Green Master Mix (Applied
Biosystems 4367659) with the primers shown in Table S4. PCR signals were detected using CFX
Connect (Bio-Rad). Arbp was used as an internal control.
Statistical Analysis. All data except for Fig. S10 are expressed as mean ± SEM. Data in Fig. S10 are expressed as box plots with whiskers from minimum to maximum, in which the band inside boxes shows median. The significance of difference was assessed by the unpaired Student’s t test, one-way
ANOVA or Mann–Whitney’s U test. The level of significance was set at P < 0.05.
Data availability. Metabolomic and proteomic data are available as Supporting Information. The microarray data of PGCs analyzed in the previous study (16) was used (GEO accession: GSE40412).
9
A N.S. N.S. N.S. 100
80
60
40
Survivalrate (%) 20
0 PGC Soma VR15 B C D
Soma_3 M1 Soma_2 PGC Soma_1 Normalized ESC PGC_3 M2 signal PGC_1 3 PGC_2 0 PC2(21.8%) ESC_2 -2 Soma ESC_1 M3 ESC_3
PC1 (53.7%) M4 ESC_2 ESC_3 ESC_1 PGC_3 PGC_2 PGC_1 Soma_1 Soma_2 Soma_3
Metabolomic analysis
E F G Soma_2 P1 PGC Soma_1 Soma_3 P2 PGC_2 Normalized signal Soma PGC_1 P3 PGC_3 4 0 PC2(20.8%) ESC_3 P4 -4 ESC ESC_2 ESC_1 P5
PC1 (55.7%) P6 ESC_3 ESC_1 ESC_2 PGC_3 PGC_1 PGC_2 Soma_3 Soma_1 Soma_2
Proteomic analysis
Fig. S1. The profile of metabolomic and proteomic analyses. (A) The survival rates of E13.5 male PGCs, Somas and VR15 ESCs used for metabolomic and proteomic analyses immediately after cell sorting. Values are plotted as mean ± standard error of the mean (SEM) of 3 biological replicates, N.S.: not significant (Student’s t-test). (B and E) Principal component analysis (PCA) of metabolomic (B) and proteomic (E) profiles of E13.5 male PGCs, Somas, and ESCs (3 biological replicates). (C and F) Hierarchical clustering of metabolomic (C) and proteomic (F) profiles of each cell type (clustering distance: Pearson, clustering method: Ward, 3 biological replicates). (D and G) K-mean clustering of statistically different metabolites (D, K = 4) and proteins (G, K = 6) (ANOVA, P < 0.05, 3 biological replicates).
10 A cluster KEGG ID Compound name cluster KEGG ID Compound name C00051 Glutathione (GSH) C01620 Threonic acid C00127 Oxidized glutathione (GSSG) C00246 Butyric acid C00029 Uridine diphosphate(UDP)-glucose C00077 Ornithine C00063 Cytidine triphosphate (CTP) C00183 L-Valine (Val) C01042 N-Acetyl-L-aspartic acid C05382 Sedoheptulose 7-phosphate (S7P) Cluster C00300 Creatine C00064 L-Glutamine (Gln) M1 C00588 Phosphorylcholine C00062 L-Arginine (Arg) PGC- C00346 O-Phosphoethanolamine C00318 L-Carnitine specific C00114 Choline C00047 L-Lysine (Lys) C00134 Putrescine C00093 Glycerol 3-phosphate (G3P) C01181 gamma-Butyrobetaine Cluster C00002 Adenosine triphosphate (ATP) C00044 Guanosine triphosphate (GTP) M3 C00078 L-Tryptophan (Trp) C00791 Creatinine PGC > C00049 L-Aspartic acid (Asp) C02614 Citramalate Soma > C00079 L-Phenylalanine (Phe) C00160 Glycolic acid ESC C00082 L-Tyrosine (Tyr) C00186 L-Lactic acid C00065 L-Serine (Ser) C06104 Adipic acid C00152 L-Asparagine (Asn) - 2-Hydroxyoctanoate C00135 L-Histidine (His) - 2-Hydroxyisobutyrate C00123 L-Leucine (Leu) C00489 Glutaric acid C00092 Glucose 6-phosphate (G6P) C00327 Citrulline C00148 L-Proline (Pro) Cluster C00197 3-Phospho-D-glycerate (3PG) C00085 Fructose 6-phosphate (F6P) M2 C05629 Phenylpropanoate C02571 L-Acetylcarnitine Soma > C01005 O-Phospho-L-serine C00245 Taurine PGC > C01089 (R)-3-Hydroxybutyric acid C00073 L-Methionine (Met) ESC C00117 D-Ribose 5-phosphate (R5P) C00043 Uridine diphosphate(UDP)-N-acetylglucosamine Cluster C02129 5-Oxohexanoic acid C00334 Gamma-Aminobutyric acid (GABA) M4 - 4-Oxopentanoate C00103 Glucose 1-phosphate (G1P) ESC- C06337 Terephthalic acid C00354 Fructose 1,6-bisphosphate (F1,6P) specific C02678 Dodecanedioic acid C00111 Dihydroxyacetone phosphate (DHAP) C08277 Sebacic acid Glycolysis-related C01013 Hydroxypropionic acid Nucleic acid synthesis-related C05984 2-Hydroxybutyric acid Amino acid-related C02656 Pimelic acid Glutathiones B Cluster P1 (ESC-specific) Cluster P4 (PGC>ESC>Soma)
Glycolysis/Gluconeogenesis etc. Nucleotide/ATP-binding etc. Nucleotide/ATP-binding etc. DNA repair etc. Oxidoreductase etc. Purine biosynthesis etc. 0 2 4 6 8 10 12 PPIase cyclophilin-type etc. Cluster P2 (ESC>PGC>Soma) Translation initiation etc.
Nucleotide/ATP-binding etc. 0 2 4 6 8 Carbon metabolism etc. Cluster P5 (PGC-specific) Aminoacyl-tRNA synthetase etc. RNA Helicase etc. Mitochondrion etc. Hydlorase/Protease etc. 0 2 4 6 8 10 12 14 16 Oxidoreductase etc. Cluster P3 (PGC>Soma>ESC) Ligase/poly(A) binding etc. Glutathione S-transferase etc. NAD etc. Oxidoreductase etc. 0 2 4 6 8 10 12 Fatty acid metabolism etc. Enrichment score Lipid biosynthesis etc. Mitochondrion etc.
0 2 4 6 Enrichment score Fig. S2. Metabolomic and proteomic profiling of E13.5 male PGCs, gonadal somatic cells (Somas), and ESCs. (A) A list of the metabolites included in each cluster classified with K-mean clustering (K = 4) (Fig. S1D). The amounts of metabolites per cell (fmol / cell; 3 biological replicates for each cell type) were obtained using capillary electrophoresis–mass spectrometry (CE-MS) and normalized with the volume of cells (nmol / mm3; Table S1 and Dataset S1). Abbreviations of metabolites are shown in this list. (B) Functional annotation of each protein cluster classified with K-mean clustering (K = 6) (Fig. S1G) using the Database for Annotation, Visualization, and Integrated Discovery (9). Experiments consisted of 3 biological replicates for each sample. 11 A Glucose HKs PGLS G6P RU5P G6PDX GPI1 Pentose phosphate pathway RPIA F6P S7P
PFKs FBPs R5P
F1,6P TALDO1 PRPSs
ALDOA PRPP PPAT DHAP GA3P TPI1 GAPDH Nucleotide PGKs synthesis 3PG ATP GTP PGAM1
ENOs CTP UTP PEP PKM
PGC > Soma, P < 0.05 Lactate Pyruvate PGC > Soma, P < 0.05, > 2 fold LDHA PGC < Soma, P < 0.05 PGC < Soma, P < 0.05, > 2 fold B Glycolysis
3PG Ser Gly Amino acids synthesis Pyruvate PDHs GSS PC DLD DLAT Asn Acetyl-CoA Glutathione metabolism GSR Asp CS GSH GSSG
OAA Citrate MDH1 ACO2 MDH2 GCLC Malate Cis-Aconitate GCLM FH1 ACO2
Fumarate TCA cycle Isocitrate IDH1 SDHs IDH3s IDH2 SDHD aKG Glu Succinate GLUD1 SUCLGs Succinyl-CoA OGDH SUCLA2 Mitochondrion OGDHL
Fig. S3. Integrated analysis of metabolomic and proteomic data between E13.5 male PGCs and Somas. (A and B) An integrated view of glycolysis-related (A) and TCA cycle-related (B) metabolic pathways comparing E13.5 male PGCs with Somas. Ellipses and rectangles show enzymes and metabolites, respectively. Orange and red indicate elements that are moderately or strongly (respectively) enriched in PGCs compared with Somas; light blue and blue indicate elements that are moderately or strongly (respectively) depleted in PGCs compared with Somas. Gray indicates elements that were undetected, or those that did not differ significantly between PGCs and Somas. For clarity, these figures exclude several metabolites and enzymes. 12 A B C Glycolysis TCA cycle OXPHOS
G6P F6P Citrate P = 0.21 NAD+ *** *** P = 0.23 P = 0.08 ** 0.6 ** 0.15 1.5 0.4 P = 0.15 3 3
3 0.3 0.4 0.10 1.0 0.2 0.2 0.05 0.5 nmol / mm / nmol
nmol / mm / nmol 0.1 nmol / mm / nmol
0.0 0.00 0.0 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC PGC Soma ESC F1,6P DHAP Succinate * * * 2.0 1.0 4.0 P = 0.34 0.8 1.5 3 3 3.0 0.6 N.S. 1.0 2.0 N.S. 0.4 0.5 nmol / mm / nmol 1.0 nmol / mm / nmol 0.2 0.0 0.0 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC GA3P 3PG Fumarate * P = 0.10 N.S. N.S. 0.10 0.20 3.0
3 0.15 3 2.0 0.05 0.10 1.0
nmol / mm / nmol 0.05
N.A. N.A. mm / nmol 0.00 0.00 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC G3P G1P ** P = 0.06 * 0.3 0.8
3 0.6 0.2 N.S. 0.4 0.1 0.2 nmol / mm / nmol 0.0 0.0 PGC Soma ESC PGC Soma ESC
Fig. S4. Quantitative analyses of metabolites in energy metabolic pathways in E13.5 male PGCs, Somas, and ESCs. (A to C) The concentrations of metabolites involved in glycolysis (A), TCA cycle (B), and OXPHOS (C). Abbreviations are as shown in Fig. S2A. N.A.: not available. Values are plotted as mean ± SEM of 3 biological replicates. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).
13 A Pentose phosphate pathway B NTPs
R5P S7P ATP GTP P = 0.16 ** 0.15 0.15 6 * * 8 ** * * 3
0.10 0.10 3 6 4 4 0.05 0.05 2
nmol / mm / nmol 2 nmol / mm / nmol 0.00 N.A. 0.00 0 0 PGC Soma ESC PGC Soma ESC PGC Soma ESC PGC Soma ESC N.S. C Amino acids CTP UTP P = 0.07 P = 0.06 Glu Gln 0.4 * 0.4 *
** 3 10 N.S. 0.3 0.3 6 **
3 8 0.2 0.2 6 4 0.1 0.1 nmol / mm / nmol 4 2 0.0 0.0 nmol / mm / nmol 2 PGC Soma ESC PGC Soma ESC 0 0 D Glutathiones PGC Soma ESC PGC Soma ESC GSSG * GSH Asp Asn *** P = 0.13 * ** 1.5 1.5 1.0 P = 0.11
10 ** 3
3 * 1.0 1.0
0.5 5 0.5 0.5 nmol / mm / nmol nmol / mm / nmol 0 0.0 0.0 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC PGC Soma ESC E Ser Gly Epigenetic regulation *** P = 0.08 + 6 * 40 SAM N.S. P = 0.24 N.S. 3 30 0.15 4 3 20 0.10 2
nmol / mm / nmol 10 0.05
0 0 mm / nmol PGC Soma ESC PGC Soma ESC 0.00 PGC Soma ESC
Fig. S5. Quantitative analyses of metabolites in biomaterial synthetic pathways in E13.5 male PGCs, Somas, and ESCs. (A to E) The concentration of metabolites involved in the pentose phosphate pathway (A), nucleotide synthesis (B), amino acid synthesis (C), glutathione metabolism (D), and epigenetic regulation (E). Abbreviations are as shown in Fig. S2A. N.A.: not available. Values are plotted as mean ± SEM of 3 biological replicates. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).
14 A E13.5 male genital ridge 1.5 P = 6.395E-13
DAPI GFP 1.0
0.5 Relative mean value mean Relative ATP5K Merge 0.0 PGC Soma n = 109 n = 66 B E13.5 male genital ridge
1.5 P = 4.581E-21
DAPI GFP 1.0
0.5 Relative mean value mean Relative GSR Merge 0.0 PGC Soma n = 60 n = 60 C E13.5 female genital ridge 1.5 P = 3.962E-17
DAPI GFP 1.0
0.5 Relative mean value mean Relative GSR Merge 0.0 PGC Soma n = 60 n = 60
Fig. S6. The expression of OXPHOS- and glutathione-related enzymes in E13.5 genital ridges, related to Fig. 3. (A) ATP5K immunostaining of E13.5 male genital ridges (left panel). The relative intensities of ATP5K fluorescence (right panel, PGC: n = 109, Soma: n = 66). (B and C) GSR immunostaining of E13.5 male (B) and female (C) genital ridges (left panel). The relative intensities of GSR fluorescence (right panel, n = 60). PGCs were detected as Oct4-deltaPE-GFP-positive cells. Experiments consisted of 2 biological replicates for each sample. Scale bar: 25 µm. Values are plotted as mean SEM (Student’s t-test).
15 A N.S. B C ** * 3.00 * 1.20 2.50 1.00 1.00)
= 2.00 0.80 mM 0.60 0 mM 2.5 mM 5.0 mM 1.50 0.40 (control = 1.00) = (control 1.00 Relative efficiency Relative 0.20 efficiency Relative 0.50 (Glucose 10 10 (Glucose 0.00 0.00 0 2.5 5.0 7.5 10 7.5 mM 10 mM Glucose conc. (mM) 2DG
D E F N.S. 1.40 1.0 * 1.20 ** * 40 0.8 35 1.00 30 0.80 PGC 25 0.6 * 0.60 DMSO 20 Soma 15 0.40 0.4
10 efficiency Relative 0.20 VR15 5 1.00) = (DMSO~72hr TUNEL positive (%) positive TUNEL 0.00 0.2 0 Survival rate (DMSO = 1.0) = (DMSO Survivalrate 0.0 DMSO Rotenone Rotenone day1 day2 day3
G N.S. *** 1.20 *** 1.00 GFP 0.80 1.00) 0.60
0.40 (Control = (Control
Relative efficiency efficiency Relative PH 0.20
0.00
Fig. S7. Effects of metabolic perturbations on E12.5 PGC reprogramming, related to Fig. 4. (A) Relative efficiency of EGC colony formation with 2DG up to or from 72 h after the initiation of culture. (B) Representative images of ALP staining of EGC colonies with glucose at 0 to 10 mM (as indicated). The 6th photo is an enlargement of the box indicated in the 5-mM plate image. Black and red arrows indicate EGC colony and non-specific staining, respectively. Scale bar: 500 µm. (C) Glucose concentration dependency of EGC formation rate (data from experiment portrayed in panel B). (D) TUNEL positive rate of E12.5 PGCs in culture with DMSO or rotenone. (E) Relative efficiency of EGC colony formation with DMSO or rotenone up to or from 72 h after the initiation of culture (left panel). Representative images of ALP staining of EGC colonies grown with DMSO or rotenone from 72 h after the initiation of culture (right panel, scale bar = 200 µm). (F) Temporal survival rate of E12.5 PGCs, Somas and VR15 ESCs with rotenone on day 1 ~ day 3 in culture. (G) Relative efficiency of EGC colony formation from E11.5 PGCs in the feeder-free culture (left panel). Representative images of Oct4-deltaPE-GFP fluorescence of EGC colonies (right panel, scale bar = 250 µm). Values are plotted as mean SEM of 3 biological replicates, N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). 16 Cyto+ A Day4 Cyto- - 2DG
0.58 % 15.13 % 18.53 % Cellcount
Venus intensity B Day4 C Day4 *** Cyto+ ** Glucose 25 0 1.25 2.5 5.0 25 1.60 P = 0.05 (mM) 1.40 1.20
Venus 1.00) 1.00
+= 0.80 PGCLC 0.60 0.40 day4 day4 0.20 (G25,cyto PH 0.00 cyto- 0 1.25 2.5 5 25
cyto+, Glucose(mM) D Day4 Cyto- Cyto+ Glucose 25 0 1.25 2.5 (mM)
0.13 % 14.86 % 9.97 % 12.72 % Cellcount
E Day2 Cyto+ Cyto+ Glucose 5.0 25 DMSO (mM) Rotenone
10.74 % 10.83 % 23.60 % 3.92 % Cellcount
Venus intensity
Fig. S8. Effects of metabolic perturbations on PGCLC induction, related to Fig. 5. (A, D, and E) Representative histograms of flow cytometry showing the rate of Blimp1-mVenus (BV) -positive cells. PGCLC induction with or without a glycolysis inhibitor, 2DG (A), PGCLC induction at various glucose concentrations (D), or PGCLC induction with or without rotenone (E). The BV-positive zone is highlighted in green. (B) Glucose concentration dependence of PGCLC induction. (C) Relative proportion of PGCLCs after 4 days of induction (data from experiment portrayed in panel B). For relevant panels: Venus: Blimp1-mVenus. PH: phase difference. Scale bar: 250 µm. Values are plotted as mean SEM of 3-4 biological replicates, **P < 0.01, ***P < 0.001 (Student’s t-test).
17 A Glucose depletion Aggregate day2
Blimp1 Stella Nanos3 Fgf5 Hoxb1 * 400 4 P = 0.080 40 P = 0.072 1.2 300 * 1.0 250 300 3 30
= 1) = 0.8 200 200 2 20 0.6 150
EpiLC 0.4 100 ( 100 1 10 0.2 P = 0.060 50 Relativeexpression 0 0 0 0.0 0 G0+ G0+ G0+ G0+ G0+ G25+ G25+ EpiLC G25+ G25+ EpiLC G25+ EpiLC EpiLC EpiLC B Glucose depletion BV-positive cells day4 Blimp1 Stella Nanos3 Fgf5 Hoxb1 * ** P = 0.086 250 80 80 2.0 15 ** 200 60 60 1.5 10 = 1) = 150 40 40 1.0 100 5 EpiLC 20 20 ( 50 0.5 N.S.
Relativeexpression 0 0 0 0.0 0 G0+ G0+ G0+ G0+ G0+ G25+ G25+ G25+ G25+ G25+ EpiLC EpiLC EpiLC EpiLC EpiLC
C Addition of 2DG Aggregate day2 Blimp1 Stella Nanos3 Fgf5 Hoxb1
250 * 5 N.S. 1.2 600 ** * 30 200 4 25 1.0 0.8 400 150 20 = 1) = 3 15 0.6 100 2 200 10 0.4 *** EpiLC ( 50 1 5 0.2 0.0 0 Relativeexpression 0 0 0 2DG 2DG 2DG cyto+ cyto+ 2DG cyto+ 2DG EpiLC EpiLC cyto+ EpiLC cyto+ EpiLC EpiLC
D Addition of 2DG BV-positive cells day4 Blimp1 Stella Nanos3 Fgf5 Hoxb1 N.S. 400 1.5 20 50 ** 80 *** * 300 40 60 15 1.0
= 1) = 30 200 40 10 20 0.5
EpiLC 20 5 ( 100 10 N.S.
Relativeexpression 0 0 0 0 0.0 2DG 2DG 2DG 2DG cyto+ cyto+ cyto+ 2DG cyto+ EpiLC EpiLC EpiLC cyto+ EpiLC EpiLC
E Addition of rotenone Aggregate day2
Blimp1 Stella Nanos3 Fgf5 Hoxb1 ** 100 1.2 10 * 1.4 350 *** 1.2 300 80 1.0 N.S. 8 1.0 250
= 1) = 0.8 60 6 0.8 200 0.6 40 4 0.6 150
EpiLC 0.4 0.4 ( 100 20 0.2 2 0.2 N.S. 50 Relativeexpression 0 0.0 0 0.0 0 EpiLC EpiLC EpiLC EpiLC EpiLC DMSO DMSO DMSO DMSO DMSO Rotenone Rotenone Rotenone Rotenone Rotenone Fig. S9. The expression changes of early PGC markers (Blimp1, Stella, Nanos3), an epiblast marker (Fgf5), and a mesoderm marker (Hoxb1) in the cell aggregates on day2 or in the purified BV-positive PGCLCs on day4. PGCLC induction in the depletion of glucose on day 2 (A) and day 4 (B), in the presence of 2DG on day 2(C) and day 4 (D), or in the presence of rotenone on day 2 (E) were shown. Values are plotted as mean SEM of 3 biological replicates. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t- test). 18 A Glycolysis B OXPHOS
* *** *** N.S. *** N.S. NormalizedValueIntensity
E9.5 E11.5 E13.5 E13.5 E9.5 E11.5 E13.5 E13.5 male female male female
PGC PGC
C NTPs Amino acids Glutathiones
Male PGCs
PSCs PGCLCs E11.5 PGCs (~E9.5 PGCs)
Female PGCs
Glycolysis
OXPHOS (male)
OXPHOS (female)
Fig. S10. (A and B) Comparative expression (transcript levels) of genes encoding energy metabolism- related enzymes in PGCs. Expression comparison of glycolysis (A, KEGG pathway: mmu00010) and OXPHOS (B, KEGG pathway: mmu00190) genes among E9.5, E11.5, and E13.5 PGCs (GEO accession: GSE40412) (16). Values are presented as box plots with whiskers from minimum to maximum. The band inside the boxes indicates the median value. N.S.: not significant, *P < 0.05, ***P < 0.001 (Mann–Whitney U test). (C) Schematic diagram summarizing metabolic changes observed in the process of cell conversion between PSCs and PGCs.
19 Table S1. Measurement of cell volumes of E13.5 male PGCs, Somas and ESCs.