Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells
- Department of Biomedical Sciences, University of Pennsylvania, School of Veterinary Medicine, United States
- Institute for Regenerative Medicine, University of Pennsylvania, United States
- Bell Research Center for Reproductive Health and Cancer, Japan
- Department of Neuroscience, Developmental and Regenerative Biology, The University of Texas at San Antonio, United States
- Texas Biomedical Research Institute, United States
- Southwest National Primate Research Center, United States
- Genomics Core, The University of Texas at San Antonio, United States
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, United States
Figures
Figure 1 with 1 supplement
Immunophenotypic characterization of pre-migratory Callithrix jacchus primordial germ cells (cjPGCs) at embryonic day (E)50.
(A) Bright field images of a cj embryo at E50 (Carnegie stage [CS]11). Scale bar, 1 mm. (B) (Left) Immunofluorescence (IF) images of the hindgut in the cj embryo as in (A) (transverse section), …
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Figure 1—source data 1
Negative control images for immunofluorescence studies.
- https://cdn.elifesciences.org/articles/82263/elife-82263-fig1-data1-v2.pdf
Figure 1—figure supplement 1
Immunophenotypic and transcriptomic characterization of marmoset embryos.
(A) Transabdominal ultrasound images of Callithrix jacchus (cj) embryos at embryonic day (E)50. (B) Immunofluorescence (IF) images of a cj embryo section (E50, CS11) stained for TFAP2C (red) and …
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Figure 1—figure supplement 1—source data 1
Differetially expressed genes (DEGs) among cell clusters in Figure 2B.
- https://cdn.elifesciences.org/articles/82263/elife-82263-fig1-figsupp1-data1-v2.xlsx
Figure 2
Single-cell transcriptome analyses of Callithrix jacchus primordial germ cells (cjPGCs) at embryonic day (E)50 (Carnegie stage [CS]11).
(A) Uniform manifold approximation and projection (UMAP), showing different cell types in cj embryos at E50. Cell clusters are annotated on the basis of marker genes. A cluster representing cjPGCs …
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Figure 2—source data 1
Differentially expressed genes (DEGs) among cell clusters in Figure 2B.
- https://cdn.elifesciences.org/articles/82263/elife-82263-fig2-data1-v2.xlsx
Figure 3 with 3 supplements
Generation of Callithrix jacchus primordial germ cell-like cells (cjPGCLCs) from cj induced pluripotent stem cells (cjiPSCs).
(A) Scheme for cjPGCLC induction. (B) BF images (top) and fluorescence-activated cell sorting (FACS) plots (bottom) for the floating aggregates of cjiPSCs induced to differentiate into cjPGCLCs. The …
Figure 3—figure supplement 1
Derivation and feeder free culture of Callithrix jacchus induced pluripotent stem cells (cjiPSCs).
(A) Unsupervised hierarchical clustering (UHC) of exome-sequenced genomic DNA derived from cjiPSCs (20201_6, 20201_7, 20201_10), hair follicles from the donor (38189), his sibling (38574), and …
Figure 3—figure supplement 2
Culture of Callithrix jacchus induced pluripotent stem cells (cjiPSCs) on a feeder layer with an inhibitor of WNT signaling.
(A) BF images of iPSCs cultured under OF, OF/IWR1, or OF/AITS conditions. Scale bars, 200 μm. (B) AP staining images of the indicated cjiPSCs. Scale bars, 200 μm. Arrows indicate areas of …
Figure 3—figure supplement 3
Induction of Callithrix jacchus primordial germ cell-like cells (cjPGCLCs) from OF, OF/IWR1, or FF cj induced pluripotent stem cells (cjiPSCs).
(A) Schematic of cjPGCLC induction. (B) BF images (inlet) and immunofluorescence (IF) images of d4 aggregates derived from OF or FF cjiPSCs stained for TFAP2C (red) and SOX17 (green), merged with …
Figure 4
2D expansion culture of Callithrix jacchus primordial germ cell-like cells (cjPGCLCs).
(A) Scheme for expansion culture of cjPGCLCs. (B) BF images of c2, c4, c6, c8, and c10 colonies of cjPGCLCs. The white dashed lines highlight colonies of cjPGCLCs. Scale bars, 200 μm. (C) …
Figure 5
Maturation of Callithrix jacchus primordial germ cell-like cells (cjPGCLCs) into a DDX4+ prospermatogonia-like state.
(A) Scheme for xrTestis culture. ALI, air-liquid interphase; xrTestis, xenogeneic reconstituted testis; mGSOs, mouse gonadal somatic cells derived from E12.5 mouse embryonic testes depleted of …
Figure 6 with 2 supplements
Transcriptome accompanying formation of Callithrix jacchus primordial germ cell-like cells (cjPGCLCs).
(A) Unsupervised hierarchical clustering (UHC) of the transcriptomes of all samples by using ward.D2. (B) Pearson correlation of samples as in (A). Highly correlated samples are encircled with red …
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Figure 6—source data 1
Top 5000 variably expressed genes among transcriptomes of different types of cells in Figure 6D.
- https://cdn.elifesciences.org/articles/82263/elife-82263-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Transcriptomic dynamics associated with Callithrix jacchus primordial germ cell-like cell (cjPGCLC) induction.
(A) Expression levels of bulk RNA-seq for all samples, normalized by log2(RPKM + 1). Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. Highly concordant …
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Figure 6—figure supplement 1—source data 1
Differentially expressed genes (DEGs) from pairwise comparisons in Figure 6—figure supplement 1D.
- https://cdn.elifesciences.org/articles/82263/elife-82263-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Dynamics of key marker gene expression associated with Callithrix jacchus primordial germ cell-like cell (cjPGCLC) induction.
Dynamics of the expression of key genes during cjPGCLC induction and expansion culture, as measured by quantitative PCR (qPCR).
Figure 7 with 2 supplements
scRNA-seq revealed lineage trajectory and gene expression dynamics during formation of Callithrix jacchus primordial germ cell-like cells (cjPGCLCs).
(A, B) Uniform manifold approximation and projection (UMAP) plots showing all cells annotated as ‘pluripotent/germ’ defined in Figure 7—figure supplement 1B, colored according to sample origin (A) …
Figure 7—figure supplement 1
Single-cell transcriptomes during Callithrix jacchus primordial germ cell-like cell (cjPGCLC) induction.
(A) Quality control analyses of all in vitro samples. (B–D) t-Distributed stochastic neighbor embedding (tSNE) plot of aggregated samples. Each dot represents a single cell, colored according to …
Figure 7—figure supplement 2
Comparison of primordial germ cell-like cells (PGCLCs) between humans and marmosets.
(A, B) Scatter plot showing differentially expressed genes (DEGs) between induced pluripotent stem cell (iPSCs) and PGCLCs in humans (A) or marmosets (B). DEGs are defined as log2 fold change above …
Figure 8
Genome-wide DNA methylation in Callithrix jacchus primordial germ cell-like cells (cjPGCLCs).
(A) Boxplot showing overall CpG DNA methylation levels. Mean DNA methylation levels (red diamonds) are labeled. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Callithrix jacchus) | 20201_6 (cjiPSC line) | This paper | Reprogrammed from PBMCs (Donor ID: 38189) | |
| Cell line (Callithrix jacchus) | 20201_7 (cjiPSC line) | This paper | Reprogrammed from PBMCs (Donor ID: 38189) | |
| Cell line (Callithrix jacchus) | 20201_10 (cjiPSCs line) | This paper | Reprogrammed from PBMCs (Donor ID: 38189) | |
| Cell line (Mus musculus) | STO | American Type Culture Collection | 1503 | Feeder for cjPGCLCs expansion |
| Biological sample (Callithrix jacchus) | 38189 | This paper | Hair follicles from the donor 38189 (sibling of 38574) | |
| Biological sample (Callithrix jacchus) | 38574 | This paper | Hair follicles from the donor 38574 (sibling of 38189) | |
| Biological sample (Callithrix jacchus) | 38921 | This paper | Hair follicles from the donor 38921 (twin sibling of 38922) | |
| Biological sample (Callithrix jacchus) | 38922 | This paper | Hair follicles from the donor 38922 (twin sibling of 38921) | |
| Biological sample (Callithrix jacchus) | 38667 | This paper | Hair follicles from the donor 38667 (twin sibling of 38668) | |
| Biological sample (Callithrix jacchus) | 38668 | This paper | Hair follicles from the donor 38668 (twin sibling of 38667) | |
| Biological sample (Callithrix jacchus) | cj embryos (E50, Carnegie stage 11) | This paper | Triplet embryos freshly isolated by hysterectomy | |
| Biological sample (Callithrix jacchus) | cj fetal testis | This paper | Freshly isolated testis from cj fetus at gestational week 19 | |
| Biological sample (Callithrix jacchus) | cj neonatal testis | This paper | Freshly isolated testis from a neonate | |
| Biological sample (Mus musculus) | Mouse fetal testicular somatic cells (E12.5) | This paper | Used for xrTestis culture | |
| Biological sample (Mus musculus) | Mouse embryonic fibroblast | This paper | Isolated from E12.5 mouse embryo, used as feeder cells for cjiPSCs culture | |
| Antibody | Anti-TFAP2C (mouse monoclonal) | Santa Cruz Biotechnology | sc-12762; RRID:AB_667770 | IF (1:200) |
| Antibody | Anti-SOX17 (goat polyclonal) | Neuromics | GT15094; RRID:AB_2195648 | IF (1:150) |
| Antibody | Anti-OCT3/4 (mouse monoclonal) | Santa Cruz Biotechnology | sc-5279; RRID:AB_628051 | IF (1:150) |
| Antibody | Anti-NANOG (goat polyclonal) | R&D Systems | AF1997; RRID:AB_355097 | IF (1:150) |
| Antibody | Anti-DDX4 (goat polyclonal) | R&D Systems | AF2030; RRID:AB_2277369 | IF (1:200) |
| Antibody | Anti-DAZL (rabbit polyclonal) | Abcam | ab34139; RRID:AB_731849 | IF (1:150) |
| Antibody | Anti-SOX2 (mouse monoclonal) | R&D Systems | MAB2018; RRID:AB_358009 | IF (1:150) |
| Antibody | Anti-LAMININ (rabbit polyclonal) | Abcam | ab11575; RRID:AB_298179 | IF (1:200) |
| Antibody | Anti-H3K9me2 (rabbit polyclonal) | MilliporeSigma | 07441; RRID:AB_11212297 | IF (1:150) |
| Antibody | Anti-H3K27me3 (rabbit polyclonal) | MilliporeSigma | 07449; RRID:AB_310624 | IF (1:150) |
| Antibody | Anti-SOX9 (rabbit polyclonal) | MilliporeSigma | AB5535; RRID:AB_2239761 | IF (1:150) |
| Antibody | Anti-PRDM1 (rabbit monoclonal) | Abcam | ab198287 | IF (1:100) |
| Antibody | Alexa Fluor 488-conjugated anti-rabbit IgG (donkey polyclonal) | Life Technologies | A21206; RRID:AB_2535792 | IF (1:500) |
| Antibody | Alexa Fluor 488-conjugated anti-mouse IgG (donkey polyclonal) | Life Technologies | A32766; RRID:AB_2762823 | IF (1:500) |
| Antibody | Alexa Fluor 568-conjugated anti-mouse IgG (donkey polyclonal) | Life Technologies | A10037; RRID:AB_2534013 | IF (1:500) |
| Antibody | Alexa Fluor 568-conjugated anti-rabbit IgG (donkey polyclonal) | Life Technologies | A10042; RRID:AB_2534017 | IF (1:500) |
| Antibody | Alexa Fluor 647-conjugated anti-goat IgG (donkey polyclonal) | Life Technologies | A21447; RRID:AB_2535864 | IF (1:500) |
| Antibody | Alexa Fluor 647-conjugated anti-rabbit IgG (donkey polyclonal) | Life Technologies | A31573; RRID:AB_2536183 | IF (1:500) |
| Antibody | Alexa Fluor 647-conjugated anti-human PDPN (rat monoclonal) | Biolegend | 337007; RRID:AB_1595538 | FACS (5 µl per test) |
| Antibody | BV421-conjugated anti-mouse CD49f (rat monoclonal) | Biolegend | 313623; RRID:AB_2562243 | FACS (5 µl per test) |
| Antibody | Alexa Fluor 488-conjugated anti-human/mouse SSEA3 (rat monoclonal) | Biolegend | 330305; RRID:AB_1279441 | FACS (5 µl per test) |
| Antibody | PE-conjugated anti-human SSEA4 (mouse monoclonal) | Biolegend | 330405; RRID:AB_1089207 | FACS (5 µl per test) |
| Antibody | Anti-SSEA1 antibody Microbeads (mouse monoclonal) | Miltenyi Biotec | 130-094-530; RRID:AB_2814656 | MACS removal of germ cells (20 µl per test) |
| Peptide, recombinant protein | Recombinant human BMP4 | R&D Systems | 314BP-010 | cjPGCLCs induction |
| Peptide, recombinant protein | Recombinant human LIF | MilliporeSigma | LIF1010 | cjPGCLCs induction |
| Peptide, recombinant protein | Recombinant human SCF | R&D Systems | 255-SC-010 | cjPGCLCs induction |
| Peptide, recombinant protein | Recombinant human EGF | R&D Systems | 236-EG | cjPGCLCs induction |
| Peptide, recombinant protein | Recombinant human/murine/rat Activin-A | R&D Systems | 338-AC | Trilineage differentiation |
| Peptide, recombinant protein | Recombinant human FLT-3 ligand | Gibco | PHC9414 | Generation of cjiPSCs lines |
| Peptide, recombinant protein | Recombinant human basic FGF | Gibco | PHG0264 | Generation of cjiPSCs lines |
| Peptide, recombinant protein | Recombinant human IL-3 | Gibco | PHC0034 | Generation of cjiPSCs lines |
| Peptide, recombinant protein | Recombinant human IL-6 | Gibco | PHC0065 | Generation of cjiPSCs lines |
| Peptide, recombinant protein | Recombinant human SCF | Gibco | PHC2111 | Generation of cjiPSCs lines |
| Peptide, recombinant protein | iMatrix-511silk (Laminin-511 E8 fragment) | TAKARA | 892021 | Feeder free cjiPSCs culture |
| Chemical compound, drug | Mitomycin C | MilliporeSigma | M0503 | Inactivation of feeder cells |
| Chemical compound, drug | Y-27632 dihydrochloride (ROCK inhibitor) | Tocris | 1254 | cjPGCLCs induction |
| Chemical compound, drug | Dorsomorphin | MilliporeSigma | P5499-5MG | Trilineage differentiation |
| Chemical compound, drug | SB431542 | MilliporeSigma | S4317 | Trilineage differentiation |
| Commercial assay or kit | CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo Fisher | A16517 | |
| Commercial assay or kit | LookOut Mycoplasma PCR Detection Kit | MilliporeSigma | MP0035-1KT | |
| Commercial assay or kit | Chromium Single Cell 3” GEM, Library & Gel Bead Kit v.3 | 10× Genomics | PN-1000092 | |
| Commercial assay or kit | SMRT-Seq HT plus kit | TAKARA | R400748 | |
| Commercial assay or kit | Pico Methyl-Seq Library Prep Kit | Zymo Research | D5455 | |
| Software, algorithm | FACSDiva software v.8.0.2 | BD Biosciences | ||
| Software, algorithm | Leica LasX c 3.7.2 | Leica | ||
| Software, algorithm | R v.4.1.0 | https://www.r-project.org | ||
| Software, algorithm | DAVID v.6.8 | https://david.ncifcrf.gov |
Additional files
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/82263/elife-82263-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/82263/elife-82263-mdarchecklist1-v2.docx
